Alkaline phosphatase agents for treatment of neurodevelopmental disorders

ABSTRACT

The present invention relates, inter alia, to compositions and methods, including therapeutic alkaline phosphatases that find use in the treatment or prevention of various neurodevelopmental diseases or disorders.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.62/679,310, filed Jun. 1, 2018, and U.S. Provisional Application No.62/615,227, filed Jan. 9, 2018, the contents of which are herebyincorporated by reference in their entireties.

TECHNICAL FIELD

The present invention relates, inter alia, to therapeutic alkalinephosphatases. The present invention further relates to compositionscomprising the therapeutic alkaline phosphatases and use of thecompositions in the treatment or prevention of neurodevelopmentaldisorders.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. The ASCII copy, created on Dec. 27, 2018, is74.1 KB in size and is named SYN-034PC_ST25.txt.

BACKGROUND

Alkaline phosphatase (“APs,” EC 3.1.3.1) is a hydrolase enzyme that canremove phosphate groups from various targets including nucleotides andproteins. Alkaline phosphatases are found in prokaryotic as well aseukaryotic organisms ranging from E. coli to mammals. In particular,mammalian APs have been shown to play important roles in gut hemostasis,mucosal barrier function, promotion of commensal bacteria, and defensefrom pathogens. Mammalian APs exert their properties by primarilytargeting LPS (a TLR4 agonist), flagellin (a TLRS agonist) and CpG DNA(a TLR9 agonist). APs also degrade intestine luminal NTPs (e.g., ATP,GTP, etc.), which promote the growth of good bacteria and reversesdysbiosis.

Neurodevelopmental disorders suffer from lack of treatments. Autism is aneurodevelopmental disorder characterized by impaired socialinteraction, verbal and non-verbal communication, and restricted andrepetitive behavior. Globally, autism is estimated to affect 21.7million people as of 2013. As of 2010, the number of people affected isestimated at about 1-2 per 1,000 worldwide. It occurs four to five timesmore often in boys than girls. About 1.5% of children in the UnitedStates (1 in 68) are diagnosed with Autism Spectrum Disorder (ASD) as of2014, a 30% increase from one in 88 in 2012. There are currently noFDA-approved treatment for autism.

Currently, there are no approved AP-based therapeutics on the market. Assuch, there remains a need for novel AP-based therapeutic compositionsfor the prevention and treatment of neurodevelopmental diseases.

SUMMARY

Accordingly, in some aspects, the present invention provides various APconstructs (“AP-based agents”) and therapeutic uses thereof. In variousembodiments, the AP-based agent is a mammalian or bacterial alkalinephosphatase. In some embodiments, the AP-based agent is a mammalianalkaline phosphatase. In an embodiment, the AP-based agent is anintestinal alkaline phosphatase. In some embodiments, the AP-based agentis a bacterial alkaline phosphatase. In some embodiments, the bacterialalkaline phosphatase has catalytic activity comparable to that of amammalian phosphatase. In some embodiments, the AP-based agent issecreted from the host cell.

In some aspects, the present invention provides methods for thetherapeutic use of an AP-based agent. In an embodiment, the presentinvention provides methods for the treatment or prevention of one ormore neurodevelopmental disorders.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the mean water consumption over the first 8 weeks of damfeeding.

FIG. 2 depicts the water consumption for the 14 days surroundingparturition.

FIG. 3 shows dam weights over the course of the first 8 weeks.

FIG. 4 shows dam weights over the course of 32 weeks.

FIG. 5 depicts the blood glucose concentrations as assayed from thevarious treatment groups after 8 weeks of feeding.

FIG. 6 depicts the weekly weights of mouse pups from each of the varioustreatment groups after having been weaned between post-natal day 20-22.

FIG. 7 depicts time (in seconds) that each of the various treatmentgroups spent in each chamber containing either a stranger mouse or anovel object during the three-chamber social interaction test. Thecontrol normal group was fed normal chow with vehicle water; thecontrol+HFD group was fed a high fat diet (HFD) with vehicle water; andthe Tx+HFD group was fed a high fat diet with water containing SYNBIAPII. The histogram shows the control normal group in the leftmost twobars, the control+HFD group in the middle two bars, and the Tx+HFD groupin the rightmost two bars.

FIG. 8 depicts time (in seconds) that each of the various treatmentgroups spent interacting with the stranger mouse or novel object in eachchamber during the three-chamber social interaction test. The controlnormal group was fed normal chow with vehicle water; the control+HFDgroup was fed a high fat diet (HFD) with vehicle water; and the Tx+HFDgroup was fed a high fat diet with water containing SYN BIAPII. Thehistogram shows the control normal group in the leftmost two bars, thecontrol+HFD group in the middle two bars, and the Tx+HFD group in therightmost two bars.

FIG. 9 depicts the number of interactions that each of the varioustreatment groups had with either the stranger mouse or novel object ineach chamber during the three-chamber social interaction test. Thecontrol normal group was fed normal chow with vehicle water; thecontrol+HFD group was fed a high fat diet (HFD) with vehicle water; andthe Tx+HFD group was fed a high fat diet with water containing SYNBIAPII. The histogram shows the control normal group in the leftmost twobars, the control+HFD group in the middle two bars, and the Tx+HFD groupin the rightmost two bars.

FIG. 10 depicts time (in seconds) that each of the various treatmentgroups spent in each chamber containing either a familiar mouse or astranger mouse during the three-chamber social interaction test. Thecontrol normal group was fed normal chow with vehicle water; thecontrol+HFD group was fed a high fat diet (HFD) with vehicle water; andthe Tx+HFD group was fed a high fat diet with water containing SYNBIAPII. The histogram shows the control normal group in the leftmost twobars, the control+HFD group in the middle two bars, and the Tx+HFD groupin the rightmost two bars.

FIG. 11 depicts time (in seconds) that each of the various treatmentgroups spent interacting with the familiar mouse or stranger mouse ineach chamber during the three-chamber social interaction test. Thecontrol normal group was fed normal chow with vehicle water; thecontrol+HFD group was fed a high fat diet (HFD) with vehicle water; andthe Tx+HFD group was fed a high fat diet with water containing SYNBIAPII. The histogram shows the control normal group in the leftmost twobars, the control+HFD group in the middle two bars, and the Tx+HFD groupin the rightmost two bars.

FIG. 12 depicts the number of interactions that each of the varioustreatment groups had with either the familiar mouse or the strangermouse in each chamber during the three-chamber social interaction test.The control normal group was fed normal chow with vehicle water; thecontrol+HFD group was fed a high fat diet (HFD) with vehicle water; andthe Tx+HFD group was fed a high fat diet with water containing SYNBIAPII. The histogram shows the control normal group in the leftmost twobars, the control+HFD group in the middle two bars, and the Tx+HFD groupin the rightmost two bars.

FIG. 13 depicts the number of entries into each chamber containingeither the familiar mouse or the stranger mouse of each of the varioustreatment groups during the three-chamber social interaction test. Thecontrol normal group was fed normal chow with vehicle water; thecontrol+HFD group was fed a high fat diet (HFD) with vehicle water; andthe Tx+HFD group was fed a high fat diet with water containing SYNBIAPII. The histogram shows the control normal group in the leftmost twobars, the control+HFD group in the middle two bars, and the Tx+HFD groupin the rightmost two bars.

FIG. 14 depicts the total distance travelled throughout testing of eachof the various treatment groups during stages 1-3 of the three-chambersocial interaction test. The control normal group was fed normal chowwith vehicle water; the control+HFD group was fed a high fat diet (HFD)with vehicle water; and the Tx+HFD group was fed a high fat diet withwater containing SYN BIAPII. Each set of histograms (according to Stage1, Stage 2, or Stage 3) shows the control normal group in the leftmostbar, the control+HFD group in the middle bar, and the Tx+HFD group inthe rightmost bar.

FIG. 15 depicts the number of interactions, either reciprocal orfollowing, during a reciprocal social interaction test for each of thevarious treatment groups. The control normal group was fed normal chowwith vehicle water; the control+HFD group was fed a high fat diet (HFD)with vehicle water; and the Tx+HFD group was fed a high fat diet withwater containing SYN BIAPII. Each set of histograms (according toReciprocal or Following) shows the control normal group in the leftmostbar, the control+HFD group in the middle bar, and the Tx+HFD group inthe rightmost bar.

FIG. 16 depicts the time (in seconds) of the interactions, eitherreciprocal or following, during a reciprocal social interaction test foreach of the various treatment groups. The control normal group was fednormal chow with vehicle water; the control+HFD group was fed a high fatdiet (HFD) with vehicle water; and the Tx+HFD group was fed a high fatdiet with water containing SYN BIAPII. Each set of histograms (accordingto Reciprocal or Following) shows the control normal group in theleftmost bar, the control+HFD group in the middle bar, and the Tx+HFDgroup in the rightmost bar.

FIG. 17 depicts the mean contact duration time (in seconds) of theinteractions, either reciprocal or following, during a reciprocal socialinteraction test for each of the various treatment groups. The controlnormal group was fed normal chow with vehicle water; the control+HFDgroup was fed a high fat diet (HFD) with vehicle water; and the Tx+HFDgroup was fed a high fat diet with water containing SYN BIAPII. Each setof histograms (according to Reciprocal or Following) shows the controlnormal group in the leftmost bar, the control+HFD group in the middlebar, and the Tx+HFD group in the rightmost bar.

FIG. 18 depicts the number of jumps exhibited by subjects during dyadictesting in each of the various treatment groups. The control normalgroup was fed normal chow with vehicle water; the control+HFD group wasfed a high fat diet (HFD) with vehicle water; and the Tx+HFD group wasfed a high fat diet with water containing SYN BIAPII. Each set ofhistograms (according to Stranger or Familiar) shows the control normalgroup in the leftmost bar, the control+HFD group in the middle bar, andthe Tx+HFD group in the rightmost bar.

DETAILED DESCRIPTION

The present invention is based, in part, on the discovery that AP-basedagents can be used in the treatment or prevention of one or moreneurodevelopmental disorders, including ASD. For instance, the presentinvention relates, in part, to use of an AP-based agent, such as,without limitation, orally administered intestinal alkaline phosphatase(IAP), to prevent ASD in the offspring of a pregnant woman at risk forhaving a child afflicted with ASD (e.g. a pregnant woman having one ormore risk factors for having a child with ASD, such as with one or moreof gastrointestinal dysbiosis, obesity, metabolic syndrome, gut-mediatedsystemic inflammation, and leaky gut).

In various embodiments, the present invention relates to the treatmentor prevention of a neurodevelopmental disorder, e.g., ASD, in an unbornchild or newly born child. For instance, in various embodiments, thepresent AP-based agent is administered to a mother or expecting mother,where the mother is afflicted with one or more of gastrointestinaldysbiosis, obesity, metabolic syndrome, gut-mediated systemicinflammation, and leaky gut and/or the mother or expecting mother has ahigh fat diet. In various embodiments, the mother or expecting mother isat risk for having a child with a neurodevelopmental disorder, e.g.,ASD, by being afflicted with one or more of gastrointestinal dysbiosis,obesity, metabolic syndrome, gut-mediated systemic inflammation, andleaky gut and/or having a high fat diet, and the present method reducesthe likelihood or severity of the neurodevelopmental disorder, e.g.,ASD, in the unborn child or newly born child of the mother or expectingmother. In various embodiments, the unborn child is in the first, orsecond, or third trimester. For example, the administration of thepresent AP-based agent occurs in the first, or second, or thirdtrimester. In various embodiments, the newly born child is less than 1,or 1, or 2, or 3, or 6, or 9 or 12, or 15, or 18, or 21, or 24 monthsold. In various embodiments, the newly born child is receiving nutritionfrom the mother via breast milk (e.g. breast feeding). For example, theadministration of the present AP-based agent occurs 1, or 1, or 2, or 3,or 6, or 9 or 12, or 15, or 18, or 21, or 24 months after delivery ofthe child.

Alkaline Phosphatase-Based Agents and Pharmaceutical Compositions

The present invention is directed, in part, to pharmaceuticalcompositions, formulations, and uses of one or more alkalinephosphatase-based agents (AP-based agents). Alkaline phosphatases aredimeric metalloenzymes that catalyze the hydrolysis of phosphate estersand dephosphoryl ate a variety of target substrates at physiological andhigher pHs. Alkaline phosphatases are found in prokaryotic as well as ineukaryotic organisms (e.g., in E. coli and mammals). IllustrativeAP-based agents that may be utilized in the present invention include,but are not limited to, intestinal alkaline phosphatase (IAP; e.g., calfIAP or bovine IAP, chicken IAP, goat IAP), placental alkalinephosphatase (PLAP), placental-like alkaline phosphatase, germ cellalkaline phosphatase (GCAP), tissue non-specific alkaline phosphatase(TNAP; which is primarily found in the liver, kidney, and bone), bonealkaline phosphatase, liver alkaline phosphatase, kidney alkalinephosphatase, bacterial alkaline phosphatase, fungal alkalinephosphatase, shrimp alkaline phosphatase, modified IAP, recombinant IAP,or any polypeptide comprising alkaline phosphatase activity.

In various embodiments, the present invention contemplates the use ofmammalian alkaline phosphatases including, but are not limited to,intestinal alkaline phosphatase (IAP), placental alkaline phosphatase(PLAP), germ cell alkaline phosphatase (GCAP), and the tissuenon-specific alkaline phosphatase (TNAP).

In some embodiments, the AP-based agent is IAP. IAP is produced in theproximal small intestine and is bound to the enterocytes via a GPIanchor. Some IAP is released into the intestinal lumen in conjunctionwith vesicles shed by the cells and as soluble protein stripped from thecells via phospholipases. The enzyme then traverses the small and largeintestine such that some active enzyme can be detected in the feces. Inan embodiment, the IAP is human IAP (hIAP). In an embodiment, the IAP iscalf IAP (cIAP), also known as bovine IAP (bIAP). There are multipleisozymes of bIAP, for example, with bIAP II and IV having higherspecific activity than bIAP I. In an embodiment, the IAP is any one ofthe clAP or bIAP isozymes (e.g., bIAP I, II, and IV). In an embodiment,the IAP is bIAP II. In another embodiment, the IAP is bIAP IV.

In various embodiments, the AP-based agent is hIAP or a variant thereof.In some embodiments, the AP-based agent is hIAP comprising the aminoacid sequence of SEQ ID NO:1 as depicted below.

HIAP SEQ ID NO: 1 1mqgpwvllll glrlqlslgv ipaeeenpaf wnrqaaeald aakklqpiqk vaknliffig 61dglgvptvta trilkgqkng klgpetplam drfpylalsk tynvdrqvpd saatataylc 121gvkanfqtig lsaaarfnqc nttrgnevis vmnrakqagk svgvvtttrv qhaspagtya 181htvnrnwysd admpasarqe gcgdiatqli snmdidvilg ggrkymfpmg tpdpeypada 241sqngirldgk nlvqewlakh qgawyvwnrt elmgasldqs vthlmglfep gdtkyeihrd 301ptldpslmem teaalrllsr nprgfylfve ggridhghhe gvayqaltea vmfddaiera 361gqltseedtl tlvtadhshv fsfggytlrg ssifglapsk aqdskaytsi lygngpgyvf 421nsgvrpdvne sesgspdygq qaavplsset hggedvavfa rgpqahlvhg vqeqsfvahv 481mafaaclepy tacdlappac ttdaahpvaa slpllagtll llgasaap

Without wishing to be bound by theory, it is believed that a cysteine atthe carboxy terminus of the AP-based agent (e.g., at position 500 of SEQID NO:1) may interfere with protein folding. Accordingly, in someembodiments, the AP-based agent includes a mutation of the cysteine(e.g., at position 500 of SEQ ID NO:1). In some embodiments, thecysteine is replaced with glycine.

In various embodiments, the AP-based agent is bIAP II or a variantthereof. In an embodiment, the bIAP II comprises the signal peptide andcarboxy terminus of bIAP I. In an embodiment, the bIAP II comprises anaspartate and position 248 (similar to bIAP IV). In an embodiment, thebIAP II comprises the amino acid sequence of SEQ ID NO: 2:

BIAP II with 248D assignment - SEQ ID NO: 2. The signal peptideand sequence past 480 are derived from bIAP I 1mqgacvllll glhlqlslgl ipaeeenpaf wnrqaaqald vakklqpiqt aaknvilflg 61dgmgvptvta trilkgqmng klgpetplam dqfpyvalsk tynvdrqvpd sagtataylc 121gvkgnyrtig vsaaarynqc nttrgnevts vinrakkagk avgvvtttrv qhaspagaya 181htvnrnwysd adlpadaqkn gcqdiaaqlv ynmdidvilg ggrmymfpeg tpdpeypdda 241svngvrkdkq nlvqewqakh qgaqyvwnrt allqaaddss vthlmglfep admkynvqqd 301htkdptlaem teaalqvlsr nprgfylfve ggridhghhd gkaymaltea imfdnaiaka 361neltseldtl ilvtadhshv fsfggytlrg tsifglapgk aldsksytsi lygngpgyal 421gggsrpdvng stseepsyrq qaavplaset hggedvavfa rgpqahlvhg vqeetfvahi 481mafagcvepy tdcnlpapat atsipdaahl aasppplall agamllllap tly

In various embodiments, the AP-based agent is bIAP IV or a variantthereof. In an embodiment, the bIAP IV comprises the amino acid sequenceof SEQ ID NO: 3:

BIAP IV SEQ ID NO: 3 1mgwacvllll glwlqlsltf ipaeeedpaf wnrgaagald vakklqpiqt aaknvilflg 61dgmgyptvta trilkgqmng klgpetplam dqfpyvalsk tynydrqvpd sagtataylc 121gvkgnyktig vsaaarynqc nttsgnevts vmnrakkagk svgyyttsry qhaspagaya 181htvnrnwysd adlpadaqty gcgdiatqly nnmdidvilg ggrmymfpeg tpdpeypydv 241nqtgyrkdkr nlygewqakh qgagyvwnrt ellqaandps vthlmglfep admkynvqqd 301ptkdptleem teaalqvlsr npqgfylfve ggridhghhe gkaymaltdt vmfdnaiaka 361neltseldtl ilatadhshv fsfggytlrg tsifglapsk asdnksytsi lygngpgyvl 421ggglrpdynd sisedpsyrq qaavplsses hggedvavfa rgpqahlvhg vgeetfvahv 481mafagcvepy tdcnlpapsg lsdaahlaas ppslallaga mllllapaly

Mammalian alkaline phosphatases are glycosylphosphatidyl-inositol (GPI)anchored proteins. They have signal peptides and are translated into thesecretory pathway. Once in the endoplasmic reticulum (ER), the proteinsare glycosylated and folded. There are two disulfide bonds as well as asingle free cysteine that is apparently not accessible on the surface.In the late ER, the carboxy terminus is removed and the GPI anchor isappended. GPI anchoring is therefore a process that occurs at thecarboxy terminus of the alkaline phosphatase. The inclusion of stopcodons at the anchor site enables secretion of biologically activeprotein (presumably the homodimer). While there is no consensussequence, the carboxy terminus includes three amino acids, termed omega,omega+1, and omega+2 which are followed by a short stretch ofhydrophilic amino acids and then a stretch of hydrophobic amino acids.Without wishing to be bound by theory, it is believed that thehydrophobicity is critical for embedding the carboxy terminus in the ERmembrane. Then an enzymatic reaction replaces the carboxy terminus withthe GPI anchor.

Within hPLAP, the GPI anchor is attached at an aspartate in thesequence, DAAH. Similarly hIAP, bIAP II, and bIAP IV also have this DAAHsequence conserved, potentially serving as the GPI anchor site.Mutational studies with hPLA indicate that preventing GPI anchoringresults in intracellular retention. In addition, mutations around theanchor site or in the hydrophobic domain either 1) prevent anchorattachment leading to intracellular retention or 2) do not block anchorattachment. Without wishing to be bound by theory, it is believed thatthe hydrophobic domain serves as a signal for GPI anchor attachment.Truncating or eliminating the hydrophobic domain leads to secretion.Finally, there is a single mutation in the hydrophobic domain that, inhPLAP, enables secretion of a protein with its hydrophobic domainintact.

In various embodiments, the AP-based agent of the invention is GPIanchored to a host cell. For example, the AP-based agent may be GPIanchored to the cell membrane of the host cell. In other embodiments,the AP-based agent of the invention is a secreted rather than ananchored protein. In some embodiments, the AP-based agent is not GPIanchored. In some embodiments, the AP-based agent may lack the GPIanchor site. In some embodiments, the AP-based agent comprises a stopcodon that is inserted immediately after the GPI anchor site. In anembodiment, the AP-based agent comprises a stop codon after theaspartate in the DAAH consensus site (e.g., at amino acid 503 of hIAPand bIAP IV or amino acid 506 of bIAP II).

HIAP with stop codon (SEQ ID NO: 4) 1mqgpwvllll glrlqlslgv ipaeeenpaf wnrqaaeald aakklqpiqk vaknliffig 61dglgvptvta trilkgqkng klgpetplam drfpylalsk tynvdrqvpd saatataylc 121gvkanfqtig lsaaarfnqc nttrgnevis vmnrakqagk svgvvtttrv qhaspagtya 181htvnrnwysd admpasarqe gcgdiatqli snmdidvilg ggrkymfpmg tpdpeypada 241sqngirldgk nlvqewlakh qgawyvwnrt elmgasldqs vthlmglfep gdtkyeihrd 301ptldpslmem teaalrllsr nprgfylfve ggridhghhe gvayqaltea vmfddaiera 361gqltseedtl tlvtadhshv fsfggytlrg ssifglapsk aqdskaytsi lygngpgyvf 421nsgvrpdvne sesgspdygq qaavplsset hggedvavfa rgpqahlvhg vqeqsfvahv 481mafaaclepy tacdlappag ttd BIAP II with stop codon (SEQ ID NO: 5) 1mqgacvllll glhlqlslgl ipaeeenpaf wnrgaagald vakklqpiqt aaknvilflg 61dgmgvptvta trilkgqmng klgpetplam dqfpyvalsk tynvdrqvpd sagtataylc 121gvkgnyrtig vsaaarynqc nttrgnevts vinrakkagk avgvvtttrv qhaspagaya 181htvnrnwysd adlpadaqkn gcgdiaaglv ynmdidvilg ggrmymfpeg tpdpeypdda 241svngvrkdkq nlvqewqakh qgagyvwnrt allqaaddss vthlmglfep admkynvqqd 301htkdptlaem teaalqvlsr nprgfylfve ggridhghhd gkaymaltea imfdnaiaka 361neltseldtl ilvtadhshv fsfggytlrg tsifglapgk aldsksytsi lygngpgyal 421gggsrpdvng stseepsyrq qaavplaset hggedvavfa rgpqahlvhg vqeetfvahi 481mafagcvepy tdcnlpapat atsipd BIAP IV with stop codon (SEQ ID NO: 6) 1mgwacvllll glwlqlsltf ipaeeedpaf wnrgaagald vakklqpiqt aaknvilflg 61dgmgvptvta trilkgqmng klgpetplam dqfpyvalsk tynvdrqvpd sagtataylc 121gvkgnyktig vsaaarynqc nttsgnevts vmnrakkagk svgvvttsry qhaspagaya 181htvnrnwysd adlpadaqty gcqdiatqlv nnmdidvilg ggrmymfpeg tpdpeypydv 241nqtgvrkdkr nlvqewqakh qgagyvwnrt ellqaandps vthlmglfep admkynvqqd 301ptkdptleem teaalqvlsr npqgfylfve ggridhghhe gkaymaltdt vmfdnaiaka 361neltseldtl ilatadhshv fsfggytlrg tsifglapsk asdnksytsi lygngpgyvl 421ggglrpdvnd sisedpsyrq qaavplsses hggedvavfa rgpqahlvhg vqeetfvahv 481mafagcvepy tdcnlpapsg lsd

In an embodiment, the AP-based agent is bIAP IV and includes a stopcodon after amino acid 508 to mimic a secreted PLAP construct asdepicted below:

BIAP IV with stop codon after amino acid 508 (SEQ ID NO: 7) 1mgwacvllll glwlqlsltf ipaeeedpaf wnrqaaqald vakklqpiqt aaknvilflg 61dgmgvptvta trilkgqmng klgpetplam dqfpyvalsk tynvdrqvpd sagtataylc 121gvkgnyktig vsaaarynqc nttsgnevts vmnrakkagk svgvvttsrv qhaspagaya 181htvnrnwysd adlpadaqty gcqdiatqlv nnmdidvilg ggrmymfpeg tpdpeypydv 241nqtgvrkdkr nlvqewqakh qgagyvwnrt ellqaandps vthlmglfep admkynvqqd 301ptkdptleem teaalqvlsr npqgfylfve ggridhghhe gkaymaltdt vmfdnaiaka 361neltseldtl ilatadhshv fsfggytlrg tsifglapsk asdnksytsi lygngpgyvl 421ggglrpdvnd sisedpsyrq qaavplsses hggedvavfa rgpqahlvhg vqeetfvahv 481mafagcvepy tdcnlpapsg lsdaahla

In various embodiments, the AP-based agent of the invention is a fusionprotein. In some embodiments, the AP-based agent comprises an alkalinephosphatase fused to a protein domain that replaces the GPI anchorsequence. In some embodiments, the alkaline phosphatase is fused to aprotein domain that promotes protein folding and/or protein purificationand/or protein dimerization and/or protein stability. In variousembodiments, the AP-based agent fusion protein has an extended serumhalf-life.

In an embodiment, the alkaline phosphatase is fused to an immunoglobulinFc domain and/or hinge region. In various embodiments, theimmunoglobulin Fc domain and/or hinge region is derived from the Fcdomain and/or hinge region of an antibody (e.g., of IgG, IgA, IgD, andIgE, inclusive of subclasses (e.g. IgG1, IgG2, IgG3, and IgG4, and IgA1and IgA2)). In an embodiment, the AP-based agent of the inventioncomprises an alkaline phosphatase fused to the hinge region and/or Fcdomain of IgG.

In various embodiments, the AP-based agent is fused to a Fc domain ofIgG comprising one or more mutations. In some embodiments, the one ormore mutations in the Fc domain of IgG function to increase serumhalf-life and longevity. In some embodiments, the Fc domain of IgGcomprises one or more mutations at amino acid residues 251-256, 285-290,308-314, 385-389 and 428-436, numbered according to the EU index as inKabat (see Kabat et al., (1991) Sequences of Proteins of ImmunologicalInterest, U.S. Public Health Service, National Institutes of Health,Washington, D.C.). In some embodiments, at least one of the amino acidsubstitutions is at amino acid residue 252, 254, 256, 309, 311, 433 or434. In an embodiment, the amino acid substitution at amino acid residue252 is a substitution with tyrosine, phenylalanine, tryptophan orthreonine. In an embodiment, the amino acid substitution at amino acidresidue 254 is a substitution with threonine. In an embodiment, theamino acid substitution at amino acid residue 256 is a substitution withserine, arginine, glutamine, glutamic acid, aspartic acid, or threonine.In an embodiment, the amino acid substitution at amino acid residue 309is a substitution with proline. In an embodiment, the amino acidsubstitution at amino acid residue 311 is a substitution with serine. Inan embodiment, the amino acid substitution at amino acid residue 385 isa substitution with arginine, aspartic acid, serine, threonine,histidine, lysine, alanine or glycine. In an embodiment, the amino acidsubstitution at amino acid residue 386 is a substitution with threonine,proline, aspartic acid, serine, lysine, arginine, isoleucine, ormethionine. In an embodiment, the amino acid substitution at amino acidresidue 387 is a substitution with arginine, proline, histidine, serine,threonine, or alanine. In an embodiment, the amino acid substitution atamino acid residue 389 is a substitution with proline, serine orasparagine. In an embodiment, the amino acid substitution at amino acidresidue 433 is a substitution with arginine, serine, isoleucine,proline, or glutamine. In an embodiment, the amino acid substitution atamino acid residue 434 is a substitution with histidine, phenylalanine,or tyrosine.

In some embodiments, the Fc domain of IgG comprises one or moremutations at amino acid residue 252, 254, 256, 433, 434, or 436. In anembodiment, the Fc domain of IgG includes a triple M252Y/S254T/T256Emutation or YTE mutation. In another embodiment, the Fc domain of IgGincludes a triple H433K/N434F/Y436H mutation or KFH mutation. In afurther embodiment, the Fc domain of IgG includes a YTE and KFH mutationin combination.

In some embodiments, the Fc domain of IgG contains one or more mutationsat amino acid residues 250, 253, 307, 310, 380, 428, 433, 434, and 435.Exemplary mutations include T250Q, M428L, T307A, E380A,1253A, H310A,M428L, H433K, N434A, N434F, N434S, and H435A. In an embodiment, the Fcdomain of IgG comprises a M428L/N434S mutation or LS mutation. Inanother embodiment, the Fc domain of IgG comprises a T250Q/M428Lmutation or QL mutation. In another embodiment, the Fc domain of IgGcomprises an N434A mutation. In another embodiment, the Fc domain of IgGcomprises a T307A/E380A/N434A mutation or AAA mutation. In anotherembodiment, the Fc domain of IgG comprises an 1253A/H310A/H435A mutationor IHH mutation. In another embodiment, the Fc domain of IgG comprises aH433K/N434F mutation. In another embodiment, the Fc domain of IgG regioncomprises a M252Y/S254T/T256E and a H433K/N434F mutation in combination.

Exemplary mutations in the Fc domain of IgG are described, for example,in Robbie, et al., Antimicrobial Agents and Chemotherapy (2013),57(12):6147-6153, Dall'Acqua et al, JBC (2006), 281(33):23514-24,Dall'Acqua et al, Journal of Immunology (2002), 169:5171-80, Ko et al.Nature (2014) 514:642-645, Grevys et al Journal of Immunology. (2015),194(11):5497-508, and U.S. Patent No. 7,083,784, the entire contents ofwhich are hereby incorporated by reference.

In various embodiments, the one or more mutations in the Fc domain ofIgG increases affinity for the neonatal Fc receptor (FcRn). In someembodiments, the one or more mutations in the Fc domain of IgG increasesaffinity for FcRn at a pH of about 6.0, about 6.1, about 6.2, about 6.3,about 6.4, or about 6.5.

In various embodiments, the alkaline phosphatase is fused to one or moreof PEG, XTENylation (e.g., as rPEG), polysialic acid (POLYXEN), albumin,elastin-like protein, elastin like protein (ELP), PAS, HAP, GLK, CTP,and transferrin. In various embodiments, the alkaline phosphatase isfused to one or more of the agents described in BioDrugs (2015)29:215-239, the entire contents of which are hereby incorporated byreference.

In an embodiment, the alkaline phosphatase is fused to a protein domain(e.g., an immunoglobulin Fc domain) via a linker to the GPI anchor site.For example, the alkaline phosphatase may be fused to a protein domainvia the aspartate at the GPI anchor sequence. The invention contemplatesthe use of a variety of linker sequences. In various embodiments, thelinker may be derived from naturally-occurring multi-domain proteins orare empirical linkers as described, for example, in Chichili et al,(2013), Protein Sci. 22(2):153-167, Chen et al, (2013), Adv Drug DelivRev. 65(10):1357-1369, the entire contents of which are herebyincorporated by reference. In some embodiments, the linker may bedesigned using linker designing databases and computer programs such asthose described in Chen et al., (2013), Adv Drug Deliv Rev.65(10):1357-1369 and Crasto et al., (2000), Protein Eng. 13(5):309-312,the entire contents of which are hereby incorporated by reference. Invarious embodiments, the linker may be functional. For example, withoutlimitation, the linker may function to improve the folding and/orstability, improve the expression, improve the pharmacokinetics, and/orimprove the bioactivity of the present AP-based agent. In anotherexample, the linker may function to target the AP-based agent to aparticular cell type or location.

In some embodiments, the linker is a polypeptide. In some embodiments,the linker is less than about 100 amino acids long. For example, thelinker may be less than about 100, about 95, about 90, about 85, about80, about 75, about 70, about 65, about 60, about 55, about 50, about45, about 40, about 35, about 30, about 25, about 20, about 19, about18, about 17, about 16, about 15, about 14, about 13, about 12, about11, about 10, about 9, about 8, about 7, about 6, about 5, about 4,about 3, or about 2 amino acids long. In some embodiments, the linker isflexible. In another embodiment, the linker is rigid.

In various embodiments, the linker is substantially comprised of glycineand serine residues (e.g. about 30%, or about 40%, or about 50%, orabout 60%, or about 70%, or about 80%, or about 90%, or about 95%, orabout 97% glycines and serines). In an embodiment, the linker sequenceis GGSGGSGGGGSGGGGS. Additional illustrative linkers include, but arenot limited to, linkers having the sequence LE, GGGGS, (GGGGS)_(n)(n=1-4), (Gly)₈, (Gly)₆, (EAAAK)_(n) (n=1-3), A(EAAAK)_(n)A (n=2-5),AEAAAKEAAAKA, A(EAAAK)₄ALEA(EAAAK)₄A, PAPAP, KESGSVSSEQLAQFRSLD,

EGKSSGSGSESKST, GSAGSAAGSGEF, and (XP)_(n), with X designating any aminoacid, e.g., Ala, Lys, or Glu. In various embodiments, the linker is GGS.

In some embodiments, the linker is a hinge region of an antibody (e.g.,of IgG, IgA, IgD, and IgE, inclusive of subclasses (e.g. IgG1, IgG2,IgG3, and IgG4, and IgA1 and IgA2)). In various embodiments, the linkeris a hinge region of an antibody (e.g., of IgG, IgA, IgD, and IgE,inclusive of subclasses (e.g. IgG1, IgG2, IgG3, and IgG4, and IgA1 andIgA2)).

The hinge region, found in IgG, IgA, IgD, and IgE class antibodies, actsas a flexible spacer, allowing the Fab portion to move freely in space.In contrast to the constant regions, the hinge domains are structurallydiverse, varying in both sequence and length among immunoglobulinclasses and subclasses. For example, the length and flexibility of thehinge region varies among the IgG subclasses. The hinge region of IgG1encompasses amino acids 216-231 and, because it is freely flexible, theFab fragments can rotate about their axes of symmetry and move within asphere centered at the first of two inter-heavy chain disulfide bridges.IgG2 has a shorter hinge than IgG1, with 12 amino acid residues and fourdisulfide bridges. The hinge region of IgG2 lacks a glycine residue, isrelatively short, and contains a rigid poly-proline double helix,stabilized by extra inter-heavy chain disulfide bridges. Theseproperties restrict the flexibility of the IgG2 molecule. IgG3 differsfrom the other subclasses by its unique extended hinge region (aboutfour times as long as the IgG1 hinge), containing 62 amino acids(including 21 prolines and 11 cysteines), forming an inflexiblepoly-proline double helix. In IgG3, the Fab fragments are relatively faraway from the Fc fragment, giving the molecule a greater flexibility.The elongated hinge in IgG3 is also responsible for its higher molecularweight compared to the other subclasses. The hinge region of IgG4 isshorter than that of IgG1 and its flexibility is intermediate betweenthat of IgG1 and IgG2. The flexibility of the hinge regions reportedlydecreases in the order IgG3>IgG1>IgG4>IgG2.

According to crystallographic studies, the immunoglobulin hinge regioncan be further subdivided functionally into three regions: the upperhinge region, the core region, and the lower hinge region. See Shin etal., 1992 Immunological Reviews 130:87. The upper hinge region includesamino acids from the carboxyl end of CH1 to the first residue in thehinge that restricts motion, generally the first cysteine residue thatforms an interchain disulfide bond between the two heavy chains. Thelength of the upper hinge region correlates with the segmentalflexibility of the antibody. The core hinge region contains theinter-heavy chain disulfide bridges, and the lower hinge region joinsthe amino terminal end of the C_(H2) domain and includes residues inC_(H2). The core hinge region of wild-type human IgG1 contains thesequence Cys-Pro-Pro-Cys which, when dimerized by disulfide bondformation, results in a cyclic octapeptide believed to act as a pivot,thus conferring flexibility. In various embodiments, the present linkercomprises, one, or two, or three of the upper hinge region, the coreregion, and the lower hinge region of any antibody (e.g., of IgG, IgA,IgD, and IgE, inclusive of subclasses (e.g. IgG1, IgG2, IgG3, and IgG4,and IgA1 and IgA2)). The hinge region may also contain one or moreglycosylation sites, which include a number of structurally distincttypes of sites for carbohydrate attachment. For example, IgA1 containsfive glycosylation sites within a 17-amino-acid segment of the hingeregion, conferring resistance of the hinge region polypeptide tointestinal proteases, considered an advantageous property for asecretory immunoglobulin. In various embodiments, the linker of thepresent invention comprises one or more glycosylation sites.

In some embodiments, the linker is a synthetic linker such as PEG.

Illustrative Fc fusion constructs of the invention include:

BIAP II with Fc Fusion - Fc domain is underlined (SEQ ID NO: 8) 1mqgacvllll glhlqlslgl ipaeeenpaf wnrgaagald vakklqpiqt aaknvilflg 61dgmgvptvta trilkgqmng klgpetplam dqfpyvalsk tynvdrqvpd sagtataylc 121gvkgnyrtig vsaaarynqc nttrgnevts vinrakkagk avgvvtttry qhaspagaya 181htvnrnwysd adlpadaqkn gcgdiaaglv ynmdidvilg ggrmymfpeg tpdpeypdda 241svngvrkdkq nlvqewqakh qgagyvwnrt allqaaddss vthlmglfep admkynvqqd 301htkdptlaem teaalqvlsr nprgfylfve ggridhghhd gkaymaltea imfdnaiaka 361neltseldtl ilvtadhshv fsfggytlrg tsifglapgk aldsksytsi lygngpgyal 421gggsrpdvng stseepsyrq qaavplaset hggedvavfa rgpqahlvhg vqeetfvahi 481mafagcvepy tdcnlpapat atsipdGGSGGSGGGGSGGGGSEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMI SRTPEVICVVVDVSHEDPQV KFNWYVDGVQVHNAKTKPREQQYNSTYRVVSVLTVLHQNW LDGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFY PSDIAVEWESNGQPENNYKT TPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALH NHYTQKSLSLSPGKBIAP IV with Fc Fusion - Fc domain is underlined (SEQ ID NO: 9) 1mgwacvllll glwlqlsltf ipaeeedpaf wnrqaaqald vakklqpiqt aaknvilflg 61dgmgvptvta trilkgqmng klgpetplam dqfpyvalsk tynvdrqvpd sagtataylc 121gvkgnyktig vsaaarynqc nttsgnevts vmnrakkagk svgvvttsry qhaspagaya 181htvnrnwysd adlpadaqty gcqdiatqlv nnmdidvilg ggrmymfpeg tpdpeypydv 241nqtgvrkdkr nlvqewqakh qgagyvwnrt ellqaandps vthlmglfep admkynvqqd 301ptkdptleem teaalqvlsr npqgfylfve ggridhghhe gkaymaltdt vmfdnaiaka 361neltseldtl ilatadhshv fsfggytlrg tsifglapsk asdnksytsi lygngpgyvl 421ggglrpdvnd sisedpsyrq qaavplsses hggedvavfa rgpqahlvhg vqeetfvahv 481mafagcvepy tdcnlpapsg lsdGGSGGSGGGGSGGGGSEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMI SRTPEVICVVVDVSHEDPQV KFNWYVDGVQVHNAKTKPREQQYNSTYRVVSVLTVLHQNW LDGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFY PSDIAVEWESNGQPENNYKT TPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALH NHYTQKSLSLSPGKHIAP with Fc Fusion - Fc domain is underlined (SEQ ID NO: 18) 1mqgpwvllll glrlqlslgv ipaeeenpaf wnrqaaeald aakklqpiqk vaknliffig 61dglgvptvta trilkgqkng klgpetplam drfpylalsk tynvdrqvpd saatataylc 121gvkanfqtig lsaaarfnqc nttrgnevis vmnrakqagk svgvvtttry qhaspagtya 181htvnrnwysd admpasarqe gcgdiatqli snmdidvilg ggrkymfpmg tpdpeypada 241sqngirldgk nlvqewlakh qgawyvwnrt elmgasldqs vthlmglfep gdtkyeihrd 301ptldpslmem teaalrllsr nprgfylfve ggridhghhe gvayqaltea vmfddaiera 361gqltseedtl tlvtadhshv fsfggytlrg ssifglapsk aqdskaytsi lygngpgyvf 421nsgvrpdvne sesgspdygq qaavplsset hggedvavfa rgpqahlvhg vqeqsfvahv 481mafaaclepy tacdlappac ttdaahpvaa slpllagtll llgasaapGGSGGSGGGGSGGGGSEPKSCDKTHTCPPCPAPE LLGGPSVFLFPPKPKDTLMISRTPEVICVVVDVSHEDPQV KFNWYVDGVQVHNAKTKPRE QQYNSTYRVVSVLTVLHQNWLDGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPP SREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKT TPPVLDSDGSFFLYSKLTVD KSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

In various embodiments, the linker can be substituted with any otherlinker disclosed herein.

A Saccharomyces alkaline phosphatase, Pho8, is produced as an inactivepro-enzyme. It is not GPI anchored, but is a transmembrane protein withits amino terminus extending out of a lysosome into the cytoplasm.Within the lysosome, an enzyme, PEP4, cleaves the carboxy terminus toactivate the enzyme. Without wishing to be bound by theory, it isbelieved that mammalian alkaline phosphatases may also be generated asinactive pro-enzymes. This is because alkaline phosphatases candephosphorylate ATP, so that activity in the ER could drain the ER ofits major energy source. Without wishing to be bound by theory, it isbelieved that the inhibitory function is located to the carboxy terminusthat would be relieved upon GPI anchor addition. Alternatively, otheractivities such as folding or metal (Zn or Mg) inclusion could controlactivity.

In various embodiments, the AP-based agent of the invention is apro-enzyme. In an embodiment, the activity of the proenzyme issuppressed by a carboxy terminus. In an embodiment, protease removal ofthe carboxy terminus reactivates the enzymatic activity of the alkalinephosphatase. In an embodiment, the pro-enzyme is more efficientlysecreted than the enzyme without the carboxy terminus.

In some embodiments, for generation of the pro-enzyme, the nativecarboxy terminus of the alkaline phosphatase is replaced with theanalogous sequence from hPLAP. In some embodiments, a mutation is madein the hydrophobic carboxy tail to promote protein secretion withoutcleavage of the carboxy terminus. In an illustrative embodiment, asingle point mutation such as a substitution of leucine with e.g.,arginine is generated in the hydrophobic carboxy terminus (e.g.allpllagtl is changed to e.g., allplragtl) to result in secretion of theenzyme without removal of the carboxy terminus.

In an embodiment, the AP-based agent is altered to include a specificenzyme cleavage site which allows subsequent removal of the carboxyterminus. In an embodiment, the AP-based agent includes a proteasecleavage site. Illustrative protease cleavage sites include, but are notlimited to, cleavage sites recognized by furin, Rhinovirus 163Cprotease, factor Xa protease, trpysin, chymotrypsin, elastase, pepsin,papain subtilisin, thermolysin, V-8 protease, submaxillaris protease,clostripain, thrombin, collagenase, and any other endoproteases. In analternative embodiment, the AP-based agent includes a cleavage siterecognized by a digestive enzyme present in the GI tract. In suchembodiments, the AP-based agent may be administered as a pro-drug thatis subsequently activated in the GI tract.

In an illustrative embodiment, the proenzyme is a proenzyme of bIAP IVhaving the following sequences:

BIAP IV with the hPLAP Carboxy Terminus and Mutation for UnprocessedSecretion and RV3C Cleavage (at ...LEVLFQGP...): SEQ ID NO: 10 1mgwacvllll glwlqlsltf ipaeeedpaf wnrgaagald vakklqpiqt aaknvilflg 61dgmgvptvta trilkgqmng klgpetplam dqfpyvalsk tynvdrqvpd sagtataylc 121gvkgnyktig vsaaarynqc nttsgnevts vmnrakkagk svgvvttsry qhaspagaya 181htvnrnwysd adlpadaqty gcqdiatqlv nnmdidvilg ggrmymfpeg tpdpeypydv 241nqtgvrkdkr nlvqewqakh qgagyvwnrt ellqaandps vthlmglfep admkynvqqd 301ptkdptleem teaalqvlsr npqgfylfve ggridhghhe gkaymaltdt vmfdnaiaka 361neltseldtl ilatadhshv fsfggytlrg tsifglapsk asdnksytsi lygngpgyvl 421ggglrpdvnd sisedpsyrq qaavplsses hggedvavfa rgpqahlvhg vqeetfvahv 481mafagcvepy tdcn

appagttd aahpgrsvvp allplragtl llletatapBIAP IV with hPLAP Carboxy Terminus and Mutation for UnprocessedSecretion and FXa Cleavage (at ...IEGR...): SEQ ID NO: 11 1mgwacvllll glwlqlsltf ipaeeedpaf wnrgaagald vakklqpiqt aaknvilflg 61dgmgvptvta trilkgqmng klgpetplam dqfpyvalsk tynvdrqvpd sagtataylc 121gvkgnyktig vsaaarynqc nttsgnevts vmnrakkagk svgvvttsry qhaspagaya 181htvnrnwysd adlpadaqty gcqdiatqlv nnmdidvilg ggrmymfpeg tpdpeypydv 241nqtgvrkdkr nlvqewqakh qgagyvwnrt ellqaandps vthlmglfep admkynvqqd 301ptkdptleem teaalqvlsr npqgfylfve ggridhghhe gkaymaltdt vmfdnaiaka 361neltseldtl ilatadhshv fsfggytlrg tsifglapsk asdnksytsi lygngpgyvl 421ggglrpdvnd sisedpsyrq qaavplsses hggedvavfa rgpqahlvhg vqeetfvahv 481mafagcvepy tdcnlappag ttdaahp

svvpallpl ragtllllet atap

In various embodiments, the AP-based agent of the invention isefficiently expressed and secreted from a host cell. In an embodiment,the AP-based agent of the invention is efficiently transcribed in thehost cell. In another embodiment, the AP-based agent exhibits enhancedRNA stability and/or transport in the host cell. In another embodiment,the AP-based agent is efficiently translated in the host cell. In afurther embodiment, the AP-based agent is efficiently folded and/ortransits efficiently through the ER, pre-golgi, and golgi. In anotherembodiment, the AP-based agent exhibits enhanced protein stability.

In various embodiments, the AP-based agent of the invention is GPIanchored to the cell membrane of a host cell. In other embodiments, theAP-based agent is secreted from the host cell. In such embodiments, theAP-based agent may include a protease cleavage site just upstream fromthe GPI anchor site. Illustrative protease cleavage sites are describedpreviously. In an embodiment, the protease cleavage site is a furincleavage site. In another embodiment, the AP-based agent may include acleavage site recognized by a digestive enzyme in the GI tract justupstream from the GPI anchor site. In these embodiments, the AP-basedagent is anchored in the ER and released in the late golgi and secreted.

In various embodiments, the AP-based agents are efficiently expressed ina host cell. In an embodiment, the Kozak sequence of the DNA constructencoding the AP-based agent is optimized. The Kozak sequence is thenucleotide sequence flanking the ATG start codon that instructs theribosome to start translation. There is flexibility in the design of aKozak sequence, but one canonical sequence is GCCGCCACCATGG. The purinein the −3 position and the G in the +4 position are the most importantbases for translation initiation. For hIAP, bIAP II, and bIAP IV, thesecond amino acid, that is, the one after the initiator methionine, isglutamine. Codons for glutamine all have a C in the first position.Thus, their Kozak sequences all have an ATGC sequence. Accordingly, invarious embodiments, the ATGC sequence is changed to ATGG. This can beachieved by changing the second amino acid to a glycine, alanine,valine, aspartate, or glutamic acid, all of whose codons have a G in thefirst position. These amino acids may be compatible with signal peptidefunction. In alternative embodiments, the entire signal peptide issubstituted for peptide having a canonical Kozak sequence and is derivedfrom a highly expressed protein such as an immunoglobulin.

In various embodiments, the signal peptide of the AP-based agent may bedeleted and/or substituted. For example, the signal peptide may bedeleted, mutated, and/or substituted (e.g., with another signal peptide)to ensure optimal protein expression.

In some embodiments, The DNA construct encoding the AP-based agent ofthe invention comprises untranslated DNA sequences. Such sequencesinclude an intron, which may be heterologous to the IAP protein ornative to the IAP protein including the native first and/or secondintron and/or a native 3′ UTR. Without wishing to be bound by theory, itis believed that include of these sequences enhance protein expressionby stabilizing the mRNA. Accordingly, in various embodiments, the DNAconstruct encoding the AP-based agent of the invention comprises the5′UTR and/or the 3′UTR.

Provided below are illustrative IAP DNA sequences with a first intronand a 3′UTR:

hIAP with native first intron (shown as bolded and underlined)SEQ ID NO: 12ATGCAGGGGCCCTGGGTGCTGCTGCTGCTGGGCCTGAGGCTACAGCTCTCCCTGGGCGTCA TCCCAG

CTGAGGAGGAGAACCCGGCCTTCTGGAACCGCCAGGCAGCTGAGGCCCTGGATGCTGCCAAGAAGCTGCAGCCCATCCAGAAGGTCGCCAAGAACCTCATCCTCTTCCTGGGCGATGGGTTGGGGGTGCCCACGGTGACAGCCACCAGGATCCTAAAGGGGCAGAAGAATGGCAAACTGGGGCCTGAGACGCCCCTGGCCATGGACCGCTTCCCATACCTGGCTCTGTCCAAGACATACAATGTGGACAGACAGGTGCCAGACAGCGCAGCCACAGCCACGGCCTACCTGTGCGGGGTCAAGGCCAACTTCCAGACCATCGGCTTGAGTGCAGCCGCCCGCTTTAACCAGTGCAACACGACACGCGGCAATGAGGTCATCTCCGTGATGAACCGGGCCAAGCAAGCAGGAAAGTCAGTAGGAGTGGTGACCACCACACGGGTGCAGCACGCCTCGCCAGCCGGCACCTACGCACACACAGTGAACCGCAACTGGTACTCAGATGCTGACATGCCTGCCTCAGCCCGCCAGGAGGGGTGCCAGGACATCGCCACTCAGCTCATCTCCAACATGGACATTGACGTGATCCTTGGCGGAGGCCGCAAGTACATGTTTCCCATGGGGACCCCAGACCCTGAGTACCCAGCTGATGCCAGCCAGAATGGAATCAGGCTGGACGGGAAGAACCTGGTGCAGGAATGGCTGGCAAAGCACCAGGGTGCCTGGTATGTGTGGAACCGCACTGAGCTCATGCAGGCGTCCCTGGACCAGTCTGTGACCCATCTCATGGGCCTCTTTGAGCCCGGAGACACGAAATATGAGATCCACCGAGACCCCACACTGGACCCCTCCCTGATGGAGATGACAGAGGCTGCCCTGCGCCTGCTGAGCAGGAACCCCCGCGGCTTCTACCTCTTTGTGGAGGGCGGCCGCATCGACCATGGTCATCATGAGGGTGTGGCTTACCAGGCACTCACTGAGGCGGTCATGTTCGACGACGCCATTGAGAGGGCGGGCCAGCTCACCAGCGAGGAGGACACGCTGACCCTCGTCACCGCTGACCACTCCCATGTCTTCTCCTTTGGTGGCTACACCTTGCGAGGGAGCTCCATCTTCGGGTTGGCCCCCAGCAAGGCTCAGGACAGCAAAGCCTACACGTCCATCCTGTACGGCAATGGCCCGGGCTACGTGTTCAACTCAGGCGTGCGACCAGACGTGAATGAGAGCGAGAGCGGGAGCCCCGATTACCAGCAGCAGGCGGCGGTGCCCCTGTCGTCCGAGACCCACGGAGGCGAAGACGTGGCGGTGTTTGCGCGCGGCCCGCAGGCGCACCTGGTGCATGGTGTGCAGGAGCAGAGCTTCGTAGCGCATGTCATGGCCTTCGCTGCCTGTCTGGAGCCCTACACGGCCTGCGACCTGGCGCCTCCCGCCTGCACCACCGACGCCGCGCACCCAGTTGCCGCGTCGCTGCCACTGCTGGCCGGGACCCTGCTGCTGCTGGGGGCGTCCGCTGCTCCCTGA hIAP with native 3′ UTR (shown as bolded and underlined)SEQ ID NO: 13ATGCAGGGGCCCTGGGTGCTGCTGCTGCTGGGCCTGAGGCTACAGCTCTCCCTGGGCGTCATCCCAGCTGAGGAGGAGAACCCGGCCTTCTGGAACCGCCAGGCAGCTGAGGCCCTGGATGCTGCCAAGAAGCTGCAGCCCATCCAGAAGGTCGCCAAGAACCTCATCCTCTTCCTGGGCGATGGGTTGGGGGTGCCCACGGTGACAGCCACCAGGATCCTAAAGGGGCAGAAGAATGGCAAACTGGGGCCTGAGACGCCCCTGGCCATGGACCGCTTCCCATACCTGGCTCTGTCCAAGACATACAATGTGGACAGACAGGTGCCAGACAGCGCAGCCACAGCCACGGCCTACCTGTGCGGGGTCAAGGCCAACTTCCAGACCATCGGCTTGAGTGCAGCCGCCCGCTTTAACCAGTGCAACACGACACGCGGCAATGAGGTCATCTCCGTGATGAACCGGGCCAAGCAAGCAGGAAAGTCAGTAGGAGTGGTGACCACCACACGGGTGCAGCACGCCTCGCCAGCCGGCACCTACGCACACACAGTGAACCGCAACTGGTACTCAGATGCTGACATGCCTGCCTCAGCCCGCCAGGAGGGGTGCCAGGACATCGCCACTCAGCTCATCTCCAACATGGACATTGACGTGATCCTTGGCGGAGGCCGCAAGTACATGTTTCCCATGGGGACCCCAGACCCTGAGTACCCAGCTGATGCCAGCCAGAATGGAATCAGGCTGGACGGGAAGAACCTGGTGCAGGAATGGCTGGCAAAGCACCAGGGTGCCTGGTATGTGTGGAACCGCACTGAGCTCATGCAGGCGTCCCTGGACCAGTCTGTGACCCATCTCATGGGCCTCTTTGAGCCCGGAGACACGAAATATGAGATCCACCGAGACCCCACACTGGACCCCTCCCTGATGGAGATGACAGAGGCTGCCCTGCGCCTGCTGAGCAGGAACCCCCGCGGCTTCTACCTCTTTGTGGAGGGCGGCCGCATCGACCATGGTCATCATGAGGGTGTGGCTTACCAGGCACTCACTGAGGCGGTCATGTTCGACGACGCCATTGAGAGGGCGGGCCAGCTCACCAGCGAGGAGGACACGCTGACCCTCGTCACCGCTGACCACTCCCATGTCTTCTCCTTTGGTGGCTACACCTTGCGAGGGAGCTCCATCTTCGGGTTGGCCCCCAGCAAGGCTCAGGACAGCAAAGCCTACACGTCCATCCTGTACGGCAATGGCCCGGGCTACGTGTTCAACTCAGGCGTGCGACCAGACGTGAATGAGAGCGAGAGCGGGAGCCCCGATTACCAGCAGCAGGCGGCGGTGCCCCTGTCGTCCGAGACCCACGGAGGCGAAGACGTGGCGGTGTTTGCGCGCGGCCCGCAGGCGCACCTGGTGCATGGTGTGCAGGAGCAGAGCTTCGTAGCGCATGTCATGGCCTTCGCTGCCTGTCTGGAGCCCTACACGGCCTGCGACCTGGCGCCTCCCGCCTGCACCACCGACGCCGCGCACCCAGTTGCCGCGTCGCTGCCACTGCTGGCCGGGACCCTGCTGCTGCTGGGGGCGTCCGCTGCTCCCTGA

bIAP IV with the first intron from bIAP I (shown as boldedand underlined) SEQ ID NO: 14ATGCAGTGGGCCTGTGTGCTGCTGCTGCTGGGCCTGTGGCTACAGCTCTCCCTCACCTTCAT CCCAG

CTGAGGAGGAAGACCCCGCCTTCTGGAACCGCCAGGCAGCCCAGGCCCTTGATGTAGCCAAGAAGTTGCAGCCGATCCAGACAGCTGCCAAGAATGTCATCCTCTTCTTGGGGGATGGGATGGGGGTGCCTACGGTGACAGCCACTCGGATCCTAAAGGGGCAGATGAATGGTAAGCTGGGACCTGAGACACCCCTGGCCATGGACCAGTTCCCATACGTGGCTCTGTCCAAGACATACAACGTGGACAGACAGGTGCCAGACAGCGCAGGCACTGCCACTGCCTACCTGTGTGGGGTCAAGGGCAACTACAAAACCATTGGTGTAAGTGCAGCCGCCCGCTACAACCAGTGCAACACAACAAGTGGCAATGAGGTCACGTCTGTGATGAACCGGGCCAAGAAAGCAGGAAAGTCAGTGGGAGTGGTGACCACCTCCAGGGTGCAGCATGCCTCCCCAGCCGGTGCTTATGCACACACGGTGAACCGAAACTGGTACTCAGATGCCGACCTGCCTGCCGATGCACAGACGTATGGCTGCCAGGACATCGCCACACAACTGGTCAACAACATGGATATTGACGTGATCCTGGGTGGAGGCCGAATGTACATGTTTCCTGAGGGGACCCCGGATCCTGAATACCCATACGATGTCAATCAGACTGGAGTCCGGAAGGACAAGCGGAATCTGGTGCAGGAGTGGCAGGCCAAGCACCAGGGAGCCCAGTATGTGTGGAACCGCACGGAGCTCCTTCAGGCAGCCAATGACCCCAGTGTAACACACCTCATGGGCCTCTTTGAGCCGGCAGACATGAAGTATAATGTTCAGCAAGACCCCACCAAGGACCCGACCCTGGAGGAGATGACGGAGGCGGCCCTGCAAGTGCTGAGCAGGAACCCCCAGGGCTTCTACCTCTTCGTGGAGGGAGGCCGCATTGACCACGGTCACCATGAAGGCAAAGCTTATATGGCACTGACTGATACAGTCATGTTTGACAATGCCATCGCCAAGGCTAACGAGCTCACTAGCGAACTGGACACGCTGATCCTTGCCACTGCAGACCACTCCCATGTCTTCTCTTTTGGTGGCTACACACTGCGTGGGACCTCCATTTTCGGTCTGGCCCCCAGCAAGGCCTCAGACAACAAGTCCTACACCTCCATCCTCTATGGCAATGGCCCTGGCTACGTGCTTGGTGGGGGCTTAAGGCCCGATGTTAATGACAGCATAAGCGAGGACCCCTCGTACCGGCAGCAGGCGGCCGTGCCCCTGTCTAGTGAGTCCCACGGGGGCGAGGACGTGGCGGTGTTCGCGCGAGGCCCGCAGGCGCACCTGGTGCACGGCGTGCAGGAGGAGACCTTCGTGGCGCACGTCATGGCCTTTGCGGGCTGCGTGGAGCCCTACACCGACTGCAATCTGCCGGCCCCCTCTGGCCTCTCCGACGCCGCGCACCTGGCGGCCAGCCCGCCTTCGCTGGCGCTGCTGGCCGGGGCGATGCTGCTGCTGCTGGCGCCTGCCT TGTACTGAbIAP IV with the 3′ UTR from bIAP I (shown as bolded and underlined)SEQ ID NO: 15ATGCAGTGGGCCTGTGTGCTGCTGCTGCTGGGCCTGTGGCTACAGCTCTCCCTCACCTTCATCCCAGCTGAGGAGGAAGACCCCGCCTTCTGGAACCGCCAGGCAGCCCAGGCCCTTGATGTAGCCAAGAAGTTGCAGCCGATCCAGACAGCTGCCAAGAATGTCATCCTCTTCTTGGGGGATGGGATGGGGGTGCCTACGGTGACAGCCACTCGGATCCTAAAGGGGCAGATGAATGGTAAGCTGGGACCTGAGACACCCCTGGCCATGGACCAGTTCCCATACGTGGCTCTGTCCAAGACATACAACGTGGACAGACAGGTGCCAGACAGCGCAGGCACTGCCACTGCCTACCTGTGTGGGGTCAAGGGCAACTACAAAACCATTGGTGTAAGTGCAGCCGCCCGCTACAACCAGTGCAACACAACAAGTGGCAATGAGGTCACGTCTGTGATGAACCGGGCCAAGAAAGCAGGAAAGTCAGTGGGAGTGGTGACCACCTCCAGGGTGCAGCATGCCTCCCCAGCCGGTGCTTATGCACACACGGTGAACCGAAACTGGTACTCAGATGCCGACCTGCCTGCCGATGCACAGACGTATGGCTGCCAGGACATCGCCACACAACTGGTCAACAACATGGATATTGACGTGATCCTGGGTGGAGGCCGAATGTACATGTTTCCTGAGGGGACCCCGGATCCTGAATACCCATACGATGTCAATCAGACTGGAGTCCGGAAGGACAAGCGGAATCTGGTGCAGGAGTGGCAGGCCAAGCACCAGGGAGCCCAGTATGTGTGGAACCGCACGGAGCTCCTTCAGGCAGCCAATGACCCCAGTGTAACACACCTCATGGGCCTCTTTGAGCCGGCAGACATGAAGTATAATGTTCAGCAAGACCCCACCAAGGACCCGACCCTGGAGGAGATGACGGAGGCGGCCCTGCAAGTGCTGAGCAGGAACCCCCAGGGCTTCTACCTCTTCGTGGAGGGAGGCCGCATTGACCACGGTCACCATGAAGGCAAAGCTTATATGGCACTGACTGATACAGTCATGTTTGACAATGCCATCGCCAAGGCTAACGAGCTCACTAGCGAACTGGACACGCTGATCCTTGCCACTGCAGACCACTCCCATGTCTTCTCTTTTGGTGGCTACACACTGCGTGGGACCTCCATTTTCGGTCTGGCCCCCAGCAAGGCCTCAGACAACAAGTCCTACACCTCCATCCTCTATGGCAATGGCCCTGGCTACGTGCTTGGTGGGGGCTTAAGGCCCGATGTTAATGACAGCATAAGCGAGGACCCCTCGTACCGGCAGCAGGCGGCCGTGCCCCTGTCTAGTGAGTCCCACGGGGGCGAGGACGTGGCGGTGTTCGCGCGAGGCCCGCAGGCGCACCTGGTGCACGGCGTGCAGGAGGAGACCTTCGTGGCGCACGTCATGGCCTTTGCGGGCTGCGTGGAGCCCTACACCGACTGCAATCTGCCGGCCCCCTCTGGCCTCTCCGACGCCGCGCACCTGGCGGCCAGCCCGCCTTCGCTGGCGCTGCTGGCCGGGGCGATGCTGCTGCTGCTGGCGCCTGCCTTGTACTGA

In various embodiments, the present invention contemplates the use ofbacterial alkaline phosphatases. In some embodiments, the AP-based agentof the invention is derived from Bacillus subtilis. Bacillus subtilis isa Gram-positive bacterium found in soil and the gastrointestinal tractof humans. Bacillus subtilis secretes high levels of proteins into theenvironment and in the human GI tract that are properly folded. Withoutwishing to be bound by theory, it is believed that Bacillus subtilissecreted proteins in the GI tract may be resistant to degradation bycommon gastrointestinal proteases. Bacillus subtilis expresses at highlevels an alkaline phosphatase multigene family. Among those isozymes,alkaline phosphatase IV is responsible for the majority of totalalkaline phosphatase expression and activity in B. subtilis. In someembodiments, the AP-based agent of the invention is derived fromBacillus licheniformis. In some embodiments, the AP-based agent of theinvention is derived from Escherichia coli.

Accordingly, in an illustrative embodiment, the AP-based agent of theinvention is derived from alkaline phosphatase IV of Bacillus subtilis.In an embodiment, the bacterial alkaline phosphatase may have thefollowing nucleotide and amino acid sequences:

Bacillus subtilis JH642 alkaline phosphatase IV,mature protein nucleotide sequence SEQ ID NO: 16AAAAAACAAGACAAAGCTGAGATCAGAAATGTCATTGTGATGATAGGCGACGGCATGGGGACGCCTTACATAAGAGCCTACCGTTCCATGAAAAATAACGGTGACACACCGAATAACCCGAAGTTAACAGAATTTGACCGGAACCTGACAGGCATGATGATGACGCATCCGGATGACCCTGACTATAATATTACAGATTCAGCAGCAGCCGGAACAGCATTAGCGACAGGCGTTAAGACATATAACAATGCAATTGGCGTCGATAAAAACGGAAAAAAAGTGAAATCTGTACTTGAAGAGGCCAAACAGCAAGGCAAGTCAACAGGGCTTGTCGCCACGTCTGAAATTAACCACGCCACTCCAGCCGCATATGGCGCCCACAATGAATCACGGAAAAACATGGACCAAATCGCCAACAGCTATATGGATGACAAGATAAAAGGCAAACATAAAATAGACGTGCTGCTCGGCGGCGGAAAATCTTATTTTAACCGCAAGAACAGAAACTTGACAAAGGAATTCAAACAAGCCGGCTACAGCTATGTGACAACTAAACAAGCATTGAAAAAAAATAAAGATCAGCAGGTGCTCGGGCTTTTCGCAGATGGAGGGCTTGCTAAAGCGCTCGACCGTGACAGTAAAACACCGTCTCTCAAAGACATGACGGTTTCAGCAATTGATCGCCTGAACCAAAATAAAAAAGGATTTTTCTTGATGGTCGAAGGGAGCCAGATTGACTGGGCGGCCCATGACAATGATACAGTAGGAGCCATGAGCGAGGTTAAAGATTTTGAACAGGCCTATAAAGCCGCGATTGAATTTGCGAAAAAAGACAAACATACACTTGTGATTGCAACTGCTGACCATACAACCGGCGGCTTTACCATTGGCGCAAACGGGGAAAAGAATTGGCACGCAGAACCGATTCTCTCCGCTAAGAAAACACCTGAATTCATGGCCAAAAAAATCAGTGAAGGCAAGCCGGTTAAAGATGTGCTCGCCCGCTATGCCAATCTGAAAGTCACATCTGAAGAAATCAAAAGCGTTGAAGCAGCTGCACAGGCTGACAAAAGCAAAGGGGCCTCCAAAGCCATCATCAAGATTTTTAATACCCGCTCCAACAGCGGATGGACGAGTACCGATCATACCGGCGAAGAAGTACCGGTATACGCGTACGGCCCCGGAAAAGAAAAATTCCGCGGATTGATTAACAATACGGACCAGGCAAACATCATATTTAAGATTTTAAA AACTGGAAAABacillus subtilis JH642 alkaline phosphatase IV,mature protein amino acid sequence SEQ ID NO: 17KKQDKAEIRNVIVMIGDGMGTPYIRAYRSMKNNGDTPNNPKLTEFDRNLTGMMMTHPDDPDYNITDSAAAGTALATGVKTYNNAIGVDKNGKKVKSVLEEAKQQGKSTGLVATSEINHATPAAYGAHNESRKNMDQIANSYMDDKIKGKHKIDVLLGGGKSYFNRKNRNLTKEFKQAGYSYVTTKQALKKNKDQQVLGLFADGGLAKALDRDSKTPSLKDMTVSAIDRLNQNKKGFFLMVEGSQIDWAAHDNDTVGAMSEVKDFEQAYKAAIEFAKKDKHTLVIATADHTTGGFTIGANGEKNWHAEPILSAKKTPEFMAKKISEGKPVKDVLARYANLKVTSEEIKSVEAAAQADKSKGASKAIIKIFNTRSNSGWTSTDHTGEEVPVYAYGPGKEKFR GLINNTDQANIIFKILKTGK

In some embodiments, the AP-based agent includes bacterial alkalinephosphatases that has one or more mutations that alter catalyticactivity. In some embodiments, the bacterial alkaline phosphatasesinclude one or more mutations such that their catalytic activity issimilar or higher than mammalian alkaline phosphatases. In someembodiments, the bacterial alkaline phosphatases include one or moremutations that alter their de-phosphorylation profile. In an embodiment,the bacterial alkaline phosphatases of the invention exhibit similarde-phosphorylation profile as mammalian alkaline phosphatases. In someembodiments, the bacterial alkaline phosphatases include one or moremutations that alter their activity at higher pH. In an embodiment, thebacterial alkaline phosphatases of the invention exhibit similaractivity at higher pH as mammalian alkaline phosphatases. In someembodiments, the bacterial alkaline phosphatases include one or moremutations that alter their metal requirements. In an embodiment, thebacterial alkaline phosphatases of the invention exhibits metalrequirements (e.g., Mg) as mammalian alkaline phosphatases.

For example, in certain embodiments, the AP-based agent of the inventionis derived from Bacillus subtilis JH642 alkaline phosphatase IV, and hasone or more mutations at positions 101, 328, A330, and 374. For example,the AP-based agent may include one or more of the following mutations:D101A, W328H, A330N and G374C.

In various embodiments, the AP-based agent of the invention comprises anucleotide sequence having at least about 60% (e.g. about 60%, or about61%, or about 62%, or about 63%, or about 64%, or about 65%, or about66%, or about 67%, or about 68%, or about 69%, or about 70%, or about71%, or about 72%, or about 73%, or about 74%, or about 75%, or about76%, or about 77%, or about 78%, or about 79%, or about 80%, or about81%, or about 82%, or about 83%, or about 84%, or about 85%, or about86%, or about 87%, or about 88%, or about 89%, or about 90%, or about91%, or about 92%, or about 93%, or about 94%, or about 95%, or about96%, or about 97%, or about 98%, or about 99%) sequence identity withany of the sequences disclosed herein.

In some embodiments, the AP-based agent of the invention comprises aamino sequence having at least about 60% (e.g. about 60%, or about 61%,or about 62%, or about 63%, or about 64%, or about 65%, or about 66%, orabout 67%, or about 68%, or about 69%, or about 70%, or about 71%, orabout 72%, or about 73%, or about 74%, or about 75%, or about 76%, orabout 77%, or about 78%, or about 79%, or about 80%, or about 81%, orabout 82%, or about 83%, or about 84%, or about 85%, or about 86%, orabout 87%, or about 88%, or about 89%, or about 90%, or about 91%, orabout 92%, or about 93%, or about 94%, or about 95%, or about 96%, orabout 97%, or about 98%, or about 99%) sequence identity with any of thesequences disclosed herein.

In various embodiments, the AP-based agent of the invention may comprisean amino acid sequence having one or more amino acid mutations relativeany of the protein sequences described herein. In some embodiments, theone or more amino acid mutations may be independently selected fromsubstitutions, insertions, deletions, and truncations.

In some embodiments, the amino acid mutations are amino acidsubstitutions, and may include conservative and/or non-conservativesubstitutions.

“Conservative substitutions” may be made, for instance, on the basis ofsimilarity in polarity, charge, size, solubility, hydrophobicity,hydrophilicity, and/or the amphipathic nature of the amino acid residuesinvolved. The 20 naturally occurring amino acids can be grouped into thefollowing six standard amino acid groups: (1) hydrophobic: Met, Ala,Val, Leu, Ile; (2) neutral hydrophilic: Cys, Ser, Thr; Asn, Gln; (3)acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues that influencechain orientation: Gly, Pro; and (6) aromatic: Trp, Tyr, Phe.

As used herein, “conservative substitutions” are defined as exchanges ofan amino acid by another amino acid listed within the same group of thesix standard amino acid groups shown above. For example, the exchange ofAsp by Glu retains one negative charge in the so modified polypeptide.In addition, glycine and proline may be substituted for one anotherbased on their ability to disrupt α-helices.

As used herein, “non-conservative substitutions” are defined asexchanges of an amino acid by another amino acid listed in a differentgroup of the six standard amino acid groups (1) to (6) shown above.

In various embodiments, the substitutions may also include non-classicalamino acids (e.g. selenocysteine, pyrrolysine, N-formylmethionineβ-alanine, GABA and δ-Aminolevulinic acid, 4-aminobenzoic acid (PABA),D-isomers of the common amino acids, 2,4-diaminobutyric acid, α-aminoisobutyric acid, 4-aminobutyric acid, Abu, 2-amino butyric acid, γ-Abu,ϵ-Ahx, 6-amino hexanoic acid, Aib, 2-amino isobutyric acid, 3-aminopropionic acid, ornithine, norleucine, norvaline, hydroxyproline,sarcosme, citrulline, homocitrulline, cysteic acid, t-butylglycine,t-butylalanine, phenylglycine, cyclohexylalanine, β-alanine,fluoro-amino acids, designer amino acids such as β methyl amino acids, Cα-methyl amino acids, N α-methyl amino acids, and amino acid analogs ingeneral).

Mutations may also be made to the nucleotide sequences of the alkalinephosphatases by reference to the genetic code, including taking intoaccount codon degeneracy. In various embodiments, the DNA constructencoding the AP-based agent is codon optimized for optimal proteinexpression in the host cell.

Mutations may be made to the AP-based agent of the invention to selectfor agents with desired characteristics. For examples, mutations may bemade to generate AP-based agents with enhanced catalytic activity orprotein stability. In various embodiments, directed evolution may beutilized to generate AP-based agents of the invention. For example,error-prone PCR and DNA shuffling may be used to identify mutations inthe bacterial alkaline phosphatases that confer enhanced activity.

In various embodiments, the AP-based agent of the invention possessesdesirable characteristics, including, for example, high specificactivity. In various embodiments, the alkaline phosphatase of theinvention possesses a specific activity of at least about 100 U/mg toabout 20,000 U/mg. In various embodiments, the alkaline phosphatase ofthe invention possesses a specific activity of at least about 100 U/mg,about 200 U/mg, about 300 U/mg, about 400 U/mg, about 500 U/mg, about600 U/mg, about 700 U/mg, about 800 U/mg, about 900 U/mg, about 1,000U/mg, about 2,000 U/mg, about 3,000 U/mg, about 4,000 U/mg, about 5,000U/mg, about 6,000 U/mg, about 7,000 U/mg, about 8,000 U/mg, about 9,000U/mg, about 10,000 U/mg, about 11,000 U/mg, about 12,000 U/mg, about13,000 U/mg, about 14,000 U/mg, about 15,000 U/mg, about 16,000 U/mg,about 17,000 U/mg, about 18,000 U/mg, about 19,000 U/mg, or about 20,000U/mg.

In various embodiments, the AP-based agent of the invention is stableand/or active in the GI tract, e.g. in one or more of the mouth,esophagus, stomach, duodenum, small intestine, duodenum, jejunum, ileum,large intestine, colon transversum, colon descendens, colon ascendens,colon sigmoidenum, cecum, and rectum. In a specific embodiment, thealkaline phosphatase is stable in the large intestine, optionallyselected from one or more of colon transversum, colon descendens, colonascendens, colon sigmoidenum and cecum. In a specific embodiment, thealkaline phosphatase is stable in the small intestine, optionallyselected from one or more of duodenum, jejunum, and ileum. In someembodiments, the alkaline phosphatase is resistant to proteases in theGI tract, including for example, the small intestine. In someembodiments, the alkaline phosphatase is substantially active at a pH ofabout 5.0 or above. For example, the alkaline phosphatase may besubstantially active at a pH about 6.0 to about 12, e.g. about 6.0, orabout 6.1, or about 6.2, or about 6.3, or about 6.4, or about 6.5, orabout 6.6, or about 6.7, or about 6.8, or about 6.9, or about 7.0, orabout 7.1, or about 7.2, or about 7.3, or about 7.4, or about 7.5, orabout 8.0, or about 8.5, or about 9.0, or about 9.5, or about 10.0, orabout 10.5, or about 11.0, or about 11.5, or about 12.0 (including, forexample, via formulation, as described herein). In some embodiments,stable refers to an enzyme that has a long enough half-life andmaintains sufficient activity for therapeutic effectiveness.

In various embodiments, the AP-based agent of the invention is stable inchyme.

In some embodiments, the AP-based agent described herein includesderivatives that are modified, i.e., by the covalent attachment of anytype of molecule to the alkaline phosphatase such that covalentattachment does not prevent the activity of the enzyme. For example, butnot by way of limitation, derivatives include alkaline phosphatases thathave been modified by, inter alia, glycosylation, lipidation,acetylation, pegylation, phosphorylation, amidation, derivatization byknown protecting/blocking groups, proteolytic cleavage, linkage to acellular ligand or other protein, etc. Any of numerous chemicalmodifications can be carried out, including, but not limited to specificchemical cleavage, acetylation, formylation, metabolic synthesis oftunicamycin, etc. Additionally, the derivative can contain one or morenon-classical amino acids. In various embodiments, the AP-based agent isglycosylated to ensure proper protein folding.

In still other embodiments, the AP-based agents of the invention may bemodified to add effector moieties such as chemical linkers, detectablemoieties such as for example fluorescent dyes, enzymes, substrates,bioluminescent materials, radioactive materials, and chemiluminescentmoieties, or functional moieties such as for example streptavidin,avidin, biotin, a cytotoxin, a cytotoxic agent, and radioactivematerials.

The AP-based agent described herein can possess a sufficiently basicfunctional group, which can react with an inorganic or organic acid, ora carboxyl group, which can react with an inorganic or organic base, toform a pharmaceutically acceptable salt. A pharmaceutically acceptableacid addition salt is formed from a pharmaceutically acceptable acid, asis well known in the art. Such salts include the pharmaceuticallyacceptable salts listed in, for example, Journal of PharmaceuticalScience, 66, 2-19 (1977) and The Handbook of Pharmaceutical Salts;Properties, Selection, and Use. P. H. Stahl and C. G. Wermuth (eds.),Verlag, Zurich (Switzerland) 2002, which are hereby incorporated byreference in their entirety.

Pharmaceutically acceptable salts include, by way of non-limitingexample, sulfate, citrate, acetate, oxalate, chloride, bromide, iodide,nitrate, bisulfate, phosphate, acid phosphate, isonicotinate, lactate,salicylate, acid citrate, tartrate, oleate, tannate, pantothenate,bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate,gluconate, glucaronate, saccharate, formate, benzoate, glutamate,methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate,camphorsulfonate, pamoate, phenylacetate, trifluoroacetate, acrylate,chlorobenzoate, dinitrobenzoate, hydroxybenzoate, methoxybenzoate,methyl benzoate, o-acetoxybenzoate, naphthalene-2-benzoate, isobutyrate,phenylbutyrate, a-hydroxybutyrate, butyne-1,4-dicarboxylate,hexyne-1,4-dicarboxylate, caprate, caprylate, cinnamate, glycollate,heptanoate, hippurate, malate, hydroxymaleate, malonate, mandelate,mesylate, nicotinate, phthalate, teraphthalate, propiolate, propionate,phenylpropionate, sebacate, suberate, p-bromobenzenesulfonate,chlorobenzenesulfonate, ethylsulfonate, 2-hydroxyethylsulfonate,methylsulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate,naphthalene-1,5-sulfonate, xylenesulfonate, and tartarate salts.

The term “pharmaceutically acceptable salt” also refers to a salt of thealkaline phosphatases having an acidic functional group, such as acarboxylic acid functional group, and a base. Suitable bases include,but are not limited to, hydroxides of alkali metals such as sodium,potassium, and lithium; hydroxides of alkaline earth metal such ascalcium and magnesium; hydroxides of other metals, such as aluminum andzinc; ammonia, and organic amines, such as unsubstituted orhydroxy-substituted mono-, di-, or tri-alkylamines, dicyclohexylamine;tributyl amine; pyridine; N-methyl, N-ethylamine; diethylamine;triethylamine; mono-, bis-, or tris-(2-OH-lower alkylamines), such asmono-; bis-, or tris-(2-hydroxyethyl)amine, 2-hydroxy-tert-butylamine,or tris-(hydroxymethyl)methylamine, N,N-di-lower alkyl-N-(hydroxyl-loweralkyl)-amines, such as N,N-dimethyl-N-(2-hydroxyethyl)amine ortri-(2-hydroxyethyl)amine; N-methyl-D-glucamine; and amino acids such asarginine, lysine, and the like.

In some embodiments, the compositions described herein are in the formof a pharmaceutically acceptable salt.

Further, any AP-based agent described herein can be administered to asubject as a component of a composition that comprises apharmaceutically acceptable carrier or vehicle. Such compositions canoptionally comprise a suitable amount of a pharmaceutically acceptableexcipient so as to provide the form for proper administration.

Pharmaceutical excipients can be liquids, such as water and oils,including those of petroleum, animal, vegetable, or synthetic origin,such as peanut oil, soybean oil, mineral oil, sesame oil and the like.The pharmaceutical excipients can be, for example, saline, gum acacia,gelatin, starch paste, talc, keratin, colloidal silica, urea and thelike. In addition, auxiliary, stabilizing, thickening, lubricating, andcoloring agents can be used. In one embodiment, the pharmaceuticallyacceptable excipients are sterile when administered to a subject. Wateris a useful excipient when any agent described herein is administeredintravenously. Saline solutions and aqueous dextrose and glycerolsolutions can also be employed as liquid excipients, specifically forinjectable solutions. Suitable pharmaceutical excipients also includestarch, glucose, cellulose, hypromellose, lactose, sucrose, malt, rice,flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc,sodium chloride, dried skim milk, glycerol, propylene, glycol, povidone,crosspovidone, water, ethanol and the like. Any agent described herein,if desired, can also comprise minor amounts of wetting or emulsifyingagents, or pH buffering agents. Other examples of suitablepharmaceutical excipients are described in Remington's PharmaceuticalSciences 1447-1676 (Alfonso R. Gennaro eds., 19th ed. 1995),incorporated herein by reference.

Where necessary, the AP-based agent and/or pharmaceutical compositions(and/or additional therapeutic agents) can include a solubilizing agent.Also, the agents can be delivered with a suitable vehicle or deliverydevice. Compositions for administration can optionally include a localanesthetic such as, for example, lignocaine to lessen pain at the siteof the injection. Combination therapies outlined herein can beco-delivered in a single delivery vehicle or delivery device.

Formulations

The present invention provides the described AP-based agent and/orpharmaceutical compositions (and/or additional therapeutic agents) invarious formulations. Any AP-based agent and/or pharmaceuticalcomposition (and/or additional therapeutic agents) described herein cantake the form of tablets, pills, pellets, capsules, capsules containingliquids, capsules containing multiparticulates, powders, solutions,emulsions, drops, suppositories, emulsions, aerosols, sprays,suspensions, delayed-release formulations, sustained-releaseformulations, controlled-release formulations, or any other formsuitable for use.

The formulations comprising the AP-based agent and/or pharmaceuticalcompositions (and/or additional therapeutic agents) may conveniently bepresented in unit dosage forms. For example, the dosage forms may beprepared by methods which include the step of bringing the therapeuticagents into association with a carrier, which constitutes one or moreaccessory ingredients. For example, the formulations are prepared byuniformly and intimately bringing the therapeutic agent into associationwith a liquid carrier, a finely divided solid carrier, or both, andthen, if necessary, shaping the product into dosage forms of the desiredformulation (e.g., wet or dry granulation, powder blends, etc., followedby press tableting)

In one embodiment, the AP-based agent (and/or additional therapeuticagents) described herein is formulated as a composition adapted for amode of administration described herein

In various embodiments, the administration the AP-based agent and/orpharmaceutical compositions (and/or additional therapeutic agents) isany one of oral, intravenous, and parenteral. For example, routes ofadministration include, but are not limited to, oral, intradermal,intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal,epidural, sublingual, intranasal, intracerebral, intravaginal,transdermal, rectally, by inhalation, or topically (e.g., to the ears,nose, eyes, or skin).

In one embodiment, the AP-based agent and/or pharmaceutical compositions(and/or additional therapeutic agents) described herein is formulated asa composition adapted for oral administration. Compositions for oraldelivery can be in the form of tablets, lozenges, aqueous or oilysuspensions, granules, powders, sprinkles, emulsions, capsules, syrups,or elixirs, for example. Orally administered compositions can compriseone or more agents, for example, sweetening agents such as fructose,aspartame or saccharin; flavoring agents such as peppermint, oil ofwintergreen, or cherry; coloring agents; and preserving agents, toprovide a pharmaceutically palatable preparation. Moreover, where intablet or pill form, the compositions can be coated to delaydisintegration to provide a sustained action over an extended period oftime. Selectively permeable membranes surrounding an osmotically activeagent driving any alkaline phosphatase (and/or additional therapeuticagents) described herein are also suitable for orally administeredcompositions. In these latter platforms, fluid from the environmentsurrounding the capsule is imbibed by the driving compound, which swellsto displace the agent or agent composition through an aperture. Thesedelivery platforms can provide an essentially zero order deliveryprofile as opposed to the spiked profiles of immediate releaseformulations. A time-delay material such as glycerol monostearate orglycerol stearate can also be useful. Oral compositions can includestandard excipients such as mannitol, lactose, starch, magnesiumstearate, sodium saccharin, cellulose, ethacrylic acid and derivativepolymers thereof, and magnesium carbonate. In one embodiment, theexcipients are of pharmaceutical grade. Suspensions, in addition to theactive compounds, may contain suspending agents such as, for example,ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitanesters, microcrystalline cellulose, aluminum metahydroxide, bentonite,agar-agar, tragacanth, etc., and mixtures thereof.

In various embodiments, the AP-based agent and/or pharmaceuticalcompositions (and/or additional therapeutic agent) are formulated assolid dosage forms such as tablets, dispersible powders, granules, andcapsules. In one embodiment, the AP-based agent and/or pharmaceuticalcompositions (and/or additional therapeutic agent) are formulated as acapsule. In another embodiment, the AP-based agent and/or pharmaceuticalcompositions (and/or additional therapeutic agent) are formulated as atablet. In yet another embodiment, the AP-based agent and/orpharmaceutical compositions (and/or additional therapeutic agent) areformulated as a soft-gel capsule. In a further embodiment, the AP-basedagent and/or pharmaceutical compositions (and/or additional therapeuticagent) are formulated as a gelatin capsule.

Dosage forms suitable for parenteral administration (e.g. intravenous,intramuscular, intraperitoneal, subcutaneous and intra-articularinjection and infusion) include, for example, solutions, suspensions,dispersions, emulsions, and the like. They may also be manufactured inthe form of sterile solid compositions (e.g. lyophilized composition),which can be dissolved or suspended in sterile injectable mediumimmediately before use. They may contain, for example, suspending ordispersing agents.

In various embodiments, the formulations of the AP-based agents mayadditionally comprise a pharmaceutically acceptable carrier orexcipient. As one skilled in the art will recognize, the formulationscan be in any suitable form appropriate for the desired use and route ofadministration.

In some dosage forms, the agents described herein are mixed with atleast one inert, pharmaceutically acceptable excipient or carrier suchas sodium citrate, dicalcium phosphate, etc., and/or a) fillers orextenders such as starches, lactose, sucrose, glucose, mannitol, silicicacid, microcrystalline cellulose, and Bakers Special Sugar, etc., b)binders such as, for example, carboxymethylcellulose, alginates,gelatin, polyvinylpyrrolidone, sucrose, acacia, polyvinyl alcohol,polyvinylpyrrolidone, methylcellulose, hydroxypropyl cellulose (HPC),and hydroxymethyl cellulose etc., c) humectants such as glycerol, etc.,d) disintegrating agents such as agar-agar, calcium carbonate, potato ortapioca starch, alginic acid, certain silicates, sodium carbonate,cross-linked polymers such as crospovidone (cross-linkedpolyvinylpyrrolidone), croscarmellose sodium (cross-linked sodiumcarboxymethylcellulose), sodium starch glycolate, etc., e) solutionretarding agents such as paraffin, etc., f) absorption accelerators suchas quaternary ammonium compounds, etc., g) wetting agents such as, forexample, cetyl alcohol and glycerol monostearate, etc., h) absorbentssuch as kaolin and bentonite clay, etc., and i) lubricants such as talc,calcium stearate, magnesium stearate, solid polyethylene glycols, sodiumlauryl sulfate, glyceryl behenate, etc., and mixtures of suchexcipients. One of skill in the art will recognize that particularexcipients may have two or more functions in the oral dosage form. Inthe case of an oral dosage form, for example, a capsule or a tablet, thedosage form may also comprise buffering agents.

The formulation can additionally include a surface active agent. Surfaceactive agents suitable for use in the present invention include, but arenot limited to, any pharmaceutically acceptable, non-toxic surfactant.Classes of surfactants suitable for use in the compositions of theinvention include, but are not limited to polyethoxylated fatty acids,PEG-fatty acid diesters, PEG-fatty acid mono- and di-ester mixtures,polyethylene glycol glycerol fatty acid esters, alcohol-oiltransesterification products, polyglycerized fatty acids, propyleneglycol fatty acid esters, mixtures of propylene glycol esters-glycerolesters, mono- and diglycerides, sterol and sterol derivatives,polyethylene glycol sorbitan fatty acid esters, polyethylene glycolalkyl ethers, sugar esters, polyethylene glycol alkyl phenols,polyoxyethylene-olyoxypropylene block copolymers, sorbitan fatty acidesters, lower alcohol fatty acid esters, ionic surfactants, and mixturesthereof. In some embodiments, compositions of the invention may compriseone or more surfactants including, but not limited to, sodium laurylsulfate, polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80,and triethyl citrate.

The formulation can also contain pharmaceutically acceptableplasticizers to obtain the desired mechanical properties such asflexibility and hardness. Such plasticizers include, but are not limitedto, triacetin, citric acid esters, triethyl citrate, phthalic acidesters, dibutyl sebacate, cetyl alcohol, polyethylene glycols,polysorbates or other plasticizers.

The formulation can also include one or more application solvents. Someof the more common solvents that can be used to apply, for example, adelayed-release coating composition include isopropyl alcohol, acetone,methylene chloride and the like.

The formulation can also include one or more alkaline materials.Alkaline material suitable for use in compositions of the inventioninclude, but are not limited to, sodium, potassium, calcium, magnesiumand aluminum salts of acids such as phosphoric acid, carbonic acid,citric acid and other aluminum/magnesium compounds. In addition thealkaline material may be selected from antacid materials such asaluminum hydroxides, calcium hydroxides, magnesium hydroxides andmagnesium oxide.

In various embodiments, the formulation can additionally includemagnesium and/or zinc. Without wishing to be bound by theory, theinclusion of magnesium and/or zinc in the formulation promotes proteinfolding (e.g., dimer formation) and bioactivity of the AP-based agent.In some embodiments, the formulation can include magnesium at aconcentration of from about 1 μM to greater than 5 mM (e.g., from about1 μM to more than 5 mM), inclusive of all ranges and valuestherebetween. In some embodiments, the formulation can include zinc at aconcentration of about 1 μM to greater than 1 mM (e.g., from about 1 μMto more than 1 mM), inclusive of all ranges and values therebetween. Invarious embodiments, the formulation of the present invention issubstantially free of metal chelators.

In various embodiments, the pH of the formulation ensures that theAP-based agent is properly folded (e.g., dimer formation) and isbioactive. In some embodiments, the formulation is maintained at a pHsuch that the amino acids which coordinate the binding of magnesiumand/or zinc within the AP-based agent are not protonated. Protonation ofsuch coordinating amino acids may lead to loss of metal ions andbioactivity and dimer disassociation. In various embodiments, the pH ofthe formulation is greater than about 6, about 6.5, about 7, about 7.5,about 8, about 8.5, about 9, about 9.5, about 10, about 10.5, about 11,about 11.5, or about 12.

Besides inert diluents, the oral compositions can also include adjuvantssuch as sweetening, flavoring, and perfuming agents.

In various embodiments, the AP-based agent and/or pharmaceuticalcompositions (and/or additional therapeutic agents) are formulated forsystemic or local delivery. In an embodiment, administration issystemic. In another embodiment, it may be desirable to administerlocally to the area in need of treatment.

Various methods may be used to formulate and/or deliver the agentsdescribed herein to a location of interest. For example, the alkalinephosphatase and/or pharmaceutical compositions (and/or additionaltherapeutic agents) described herein may be formulated for delivery tothe gastrointestinal tract. The gastrointestinal tract includes organsof the digestive system such as mouth, esophagus, stomach, duodenum,small intestine, large intestine and rectum and includes all subsectionsthereof (e.g. the small intestine may include the duodenum, jejunum andileum; the large intestine may include the colon transversum, colondescendens, colon ascendens, colon sigmoidenum and cecum). For example,the alkaline phosphatases and/or pharmaceutical compositions (and/oradditional therapeutic agents) described herein may be formulated fordelivery to one or more of the stomach, small intestine, large intestineand rectum and includes all subsections thereof (e.g. duodenum, jejunumand ileum, colon transversum, colon descendens, colon ascendens, colonsigmoidenum and cecum). In some embodiments, the compositions describedherein may be formulated to deliver to the upper or lower GI tract. Inan embodiment, the alkaline phosphatases and/or pharmaceuticalcompositions (and/or additional therapeutic agents) may be administeredto a subject, by, for example, directly or indirectly contacting themucosal tissues of the gastrointestinal tract.

In various embodiments, the administration the AP-based agent and/orpharmaceutical compositions (and/or additional therapeutic agents) isinto the GI tract via, for example, oral delivery, nasogastral tube,intestinal intubation (e.g. an enteral tube or feeding tube such as, forexample, a jejunal tube or gastro-jejunal tube, etc.), direct infusion(e.g., duodenal infusion), endoscopy, colonoscopy, or enema.

For example, in various embodiments, the present invention providesmodified release formulations comprising at least one AP-based agent(and/or additional therapeutic agents), wherein the formulation releasesa substantial amount of the AP-based agent (and/or additionaltherapeutic agents) into one or more regions of the GI tract. Forexample, the formulation may release at least about 60% of the AP-basedagent after the stomach and into one or more regions of the GI tract.

In various embodiments, the modified-release formulation of the presentinvention releases at least 60% of the AP-based agent (or additionaltherapeutic agents) after the stomach into one or more regions of theintestine. For example, the modified-release formulation releases atleast 60%, at least 61%, at least 62%, at least 63%, at least 64%, atleast 65%, at least 66%, at least 67%, at least 68%, at least 69%, atleast 70%, at least 71%, at least 72%, at least 73%, at least 74%, atleast 75%, at least 76%, at least 77%, at least 78%, at least 79%, atleast 80%, at least 81%, at least 82%, at least 83%, at least 84%, atleast 85%, at least 86%, at least 87%, at least 88%, at least 89%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%, or100% of the AP-based agent (or additional therapeutic agents) in theintestines.

In various embodiments, the modified-release formulation of the presentinvention releases at least 60% of the AP-based agent (or additionaltherapeutic agents) in the small intestine. For example, themodified-release formulation releases at least 60%, at least 61%, atleast 62%, at least 63%, at least 64%, at least 65%, at least 66%, atleast 67%, at least 68%, at least 69%, at least 70%, at least 71%, atleast 72%, at least 73%, at least 74%, at least 75%, at least 76%, atleast 77%, at least 78%, at least 79%, at least 80%, at least 81%, atleast 82%, at least 83%, at least 84%, at least 85%, at least 86%, atleast 87%, at least 88%, at least 89%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or 100% of the AP-based agent (oradditional therapeutic agents) in the small intestine (e.g., one or moreof duodenum, jejunum, ileum, and ileocecal junction).

In various embodiments, the modified-release formulation of the presentinvention releases at least 60% of the AP-based agent (or additionaltherapeutic agents) in the large intestine. For example, themodified-release formulation releases at least 60%, at least 61%, atleast 62%, at least 63%, at least 64%, at least 65%, at least 66%, atleast 67%, at least 68%, at least 69%, at least 70%, at least 71%, atleast 72%, at least 73%, at least 74%, at least 75%, at least 76%, atleast 77%, at least 78%, at least 79%, at least 80%, at least 81%, atleast 82%, at least 83%, at least 84%, at least 85%, at least 86%, atleast 87%, at least 88%, at least 89%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or 100% of the AP-based agent (oradditional therapeutic agents) in the large intestine (e.g., one or moreof cecum, ascending, transverse, descending or sigmoid portions of thecolon, and rectum).

In various embodiments, the modified-release formulation does notsubstantially release the AP-based agent (or additional therapeuticagents) in the stomach.

In certain embodiments, the modified-release formulation releases theAP-based agent (or additional therapeutic agents) at a specific pH. Forexample, in some embodiments, the modified-release formulation issubstantially stable in an acidic environment and substantially unstable(e.g., dissolves rapidly or is physically unstable) in a near neutral toalkaline environment. In some embodiments, stability is indicative ofnot substantially releasing while instability is indicative ofsubstantially releasing. For example, in some embodiments, themodified-release formulation is substantially stable at a pH of about7.0 or less, or about 6.5 or less, or about 6.0 or less, or about 5.5 orless, or about 5.0 or less, or about 4.5 or less, or about 4.0 or less,or about 3.5 or less, or about 3.0 or less, or about 2.5 or less, orabout 2.0 or less, or about 1.5 or less, or about 1.0 or less. In someembodiments, the present formulations are stable in lower pH areas andtherefore do not substantially release in, for example, the stomach. Insome embodiments, modified-release formulation is substantially stableat a pH of about 1 to about 4 or lower and substantially unstable at pHvalues that are greater. In these embodiments, the modified-releaseformulation does not substantially release in the stomach. In theseembodiments, the modified-release formulation substantially releases inthe small intestine (e.g. one or more of the duodenum, jejunum, andileum) and/or large intestine (e.g. one or more of the cecum, ascendingcolon, transverse colon, descending colon, and sigmoid colon). In someembodiments, modified-release formulation is substantially stable at apH of about 4 to about 5 or lower and consequentially is substantiallyunstable at pH values that are greater and therefore is notsubstantially released in the stomach and/or small intestine (e.g. oneor more of the duodenum, jejunum, and ileum). In these embodiments, themodified-release formulation substantially releases in the largeintestine (e.g. one or more of the cecum, ascending colon, transversecolon, descending colon, and sigmoid colon). In various embodiments, thepH values recited herein may be adjusted as known in the art to accountfor the state of the subject, e.g. whether in a fasting or postprandialstate.

In some embodiments, the modified-release formulation is substantiallystable in gastric fluid and substantially unstable in intestinal fluidand, accordingly, is substantially released in the small intestine (e.g.one or more of the duodenum, jejunum, and ileum) and/or large intestine(e.g. one or more of the cecum, ascending colon, transverse colon,descending colon, and sigmoid colon).

In some embodiments, the modified-release formulation is stable ingastric fluid or stable in acidic environments. These modified-releaseformulations release about 30% or less by weight of the alkalinephosphatase and/or additional therapeutic agent in the modified-releaseformulation in gastric fluid with a pH of about 4 to about 5 or less, orsimulated gastric fluid with a pH of about 4 to about 5 or less, inabout 15, or about 30, or about 45, or about 60, or about 90 minutes.Modified-release formulations of the of the invention may release fromabout 0% to about 30%, from about 0% to about 25%, from about 0% toabout 20%, from about 0% to about 15%, from about 0% to about 10%, about5% to about 30%, from about 5% to about 25%, from about 5% to about 20%,from about 5% to about 15%, from about 5% to about 10% by weight of thealkaline phosphatase and/or additional therapeutic agent in themodified-release formulation in gastric fluid with a pH of 4-5, or lessor simulated gastric fluid with a pH of 4-5 or less, in about 15, orabout 30, or about 45, or about 60, or about 90 minutes.Modified-release formulations of the invention may release about 1%,about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%,about 9%, or about 10% by weight of the total alkaline phosphataseand/or additional therapeutic agent in the modified-release formulationin gastric fluid with a pH of 5 or less, or simulated gastric fluid witha pH of 5 or less, in about 15, or about 30, or about 45, or about 60,or about 90 minutes.

In some embodiments, the modified-release formulation is unstable inintestinal fluid. These modified-release formulations release about 70%or more by weight of the alkaline phosphatase and/or additionaltherapeutic agent in the modified-release formulation in intestinalfluid or simulated intestinal fluid in about 15, or about 30, or about45, or about 60, or about 90 minutes. In some embodiments, themodified-release formulation is unstable in near neutral to alkalineenvironments. These modified-release formulations release about 70% ormore by weight of the alkaline phosphatase and/or additional therapeuticagent in the modified-release formulation in intestinal fluid with a pHof about 4-5 or greater, or simulated intestinal fluid with a pH ofabout 4-5 or greater, in about 15, or about 30, or about 45, or about60, or about 90 minutes. A modified-release formulation that is unstablein near neutral or alkaline environments may release 70% or more byweight of alkaline phosphatase and/or additional therapeutic agent inthe modified-release formulation in a fluid having a pH greater thanabout 5 (e.g., a fluid having a pH of from about 5 to about 14, fromabout 6 to about 14, from about 7 to about 14, from about 8 to about 14,from about 9 to about 14, from about 10 to about 14, or from about 11 toabout 14) in from about 5 minutes to about 90 minutes, or from about 10minutes to about 90 minutes, or from about 15 minutes to about 90minutes, or from about 20 minutes to about 90 minutes, or from about 25minutes to about 90 minutes, or from about 30 minutes to about 90minutes, or from about 5 minutes to about 60 minutes, or from about 10minutes to about 60 minutes, or from about 15 minutes to about 60minutes, or from about 20 minutes to about 60 minutes, or from about 25minutes to about 90 minutes, or from about 30 minutes to about 60minutes.

Examples of simulated gastric fluid and simulated intestinal fluidinclude, but are not limited to, those disclosed in the 2005Pharmacopeia 23NF/28USP in Test Solutions at page 2858 and/or othersimulated gastric fluids and simulated intestinal fluids known to thoseof skill in the art, for example, simulated gastric fluid and/orintestinal fluid prepared without enzymes.

In various embodiments, the modified-release formulation of theinvention is substantially stable in chyme. For example, there is, insome embodiments, a loss of less than about 50% or about 40%, or about30%, or about 20%, or about 10% of AP-based agent activity in about 10,or 9, or 8, or 7, or 6, or 5, or 4, or 3, or 2, or 1 hour fromadministration.

In various embodiments, the modified-release formulations of the presentinvention are designed for immediate release (e.g. upon ingestion). Invarious embodiments, the modified-release formulations may havesustained-release profiles, i.e. slow release of the activeingredient(s) in the body (e.g., GI tract) over an extended period oftime. In various embodiments, the modified-release formulations may havea delayed-release profile, i.e. not immediately release the activeingredient(s) upon ingestion; rather, postponement of the release of theactive ingredient(s) until the composition is lower in thegastrointestinal tract; for example, for release in the small intestine(e.g., one or more of duodenum, jejunum, ileum) or the large intestine(e.g., one or more of cecum, ascending, transverse, descending orsigmoid portions of the colon, and rectum). For example, a compositioncan be enteric coated to delay release of the active ingredient(s) untilit reaches the small intestine or large intestine.

In various embodiments, the modified-release formulation of the presentinvention may utilize one or more modified-release coatings such asdelayed-release coatings to provide for effective, delayed yetsubstantial delivery of the alkaline phosphatase to the GI tracttogether with, optionally, additional therapeutic agents.

In various embodiments, the modified-release formulation of the presentinvention may utilize one or more modified-release coatings such asdelayed-release coatings to provide for effective, delayed yetsubstantial delivery of the alkaline phosphatase to the intestinestogether with, optionally, other additional therapeutic agents.

In one embodiment, the delayed-release coating includes an enteric agentthat is substantially stable in acidic environments and substantiallyunstable in near neutral to alkaline environments. In an embodiment, thedelayed-release coating contains an enteric agent that is substantiallystable in gastric fluid. The enteric agent can be selected from, forexample, solutions or dispersions of methacrylic acid copolymers,cellulose acetate phthalate, hydroxypropylmethyl cellulose phthalate,polyvinyl acetate phthalate, carboxymethylethylcellulose, andEUDRAGIT®-type polymer (poly(methacrylic acid, methylmethacrylate),hydroxypropyl methylcellulose acetate succinate, cellulose acetatetrimellitate, shellac or other suitable enteric coating polymers. TheEUDRAGIT®-type polymers include, for example, EUDRAGIT® FS 30D, L 30D-55, L 100-55, L 100, L 12,5, L 12,5 P, RL 30 D, RL P0, RL 100, RL12,5, RS 30 D, RS P0, RS 100, RS 12,5, NE 30 D, NE 40 D, NM 30 D, S 100,S 12,5, and S 12,5 P. Similar polymers include Kollicoat® MAE 30 DP andKollicoat® MAE 100 P. In some embodiments, one or more of EUDRAGIT® FS30D, L 30 D-55, L 100-55, L 100, L 12,5, L 12,5 P RL 30 D, RL P0, RL100, RL 12,5, RS 30 D, RS P0, RS 100, RS 12,5, NE 30 D, NE 40 D, NM 30D, S 100, S 12,5 S 12,5 P, Kollicoat® MAE 30 DP and Kollicoat® MAE 100 Pis used. In various embodiments, the enteric agent may be a combinationof the foregoing solutions or dispersions. In an embodiment, thedelayed-release coating includes the enteric agent EUDRAGIT® L 30 D-55.

In certain embodiments, one or more coating system additives are usedwith the enteric agent. For example, one or more PIasACRYL™ additivesmay be used as an anti-tacking agent coating additive. IllustrativePIasACRYL™ TM additives include, but are not limited to PIasACRYL™ HTP20and PIasACRYL™ T20. In an embodiment, PIasACRYL™ HTP20 is formulatedwith EUDRAGIT® L 30 D-55 coatings. In another embodiment, PIasACRYL™ TMT20 is formulated with EUDRAGIT® FS 30 D coatings.

In another embodiment, the delayed-release coating may degrade as afunction of time when in aqueous solution without regard to the pHand/or presence of enzymes in the solution. Such a coating may comprisea water insoluble polymer. Its solubility in aqueous solution istherefore independent of the pH. The term “pH independent” as usedherein means that the water permeability of the polymer and its abilityto release pharmaceutical ingredients is not a function of pH and/or isonly very slightly dependent on pH. Such coatings may be used toprepare, for example, sustained release formulations. Suitable waterinsoluble polymers include pharmaceutically acceptable non-toxicpolymers that are substantially insoluble in aqueous media, e.g., water,independent of the pH of the solution. Suitable polymers include, butare not limited to, cellulose ethers, cellulose esters, or celluloseether-esters, i.e., a cellulose derivative in which some of the hydroxygroups on the cellulose skeleton are substituted with alkyl groups andsome are modified with alkanoyl groups. Examples include ethylcellulose, acetyl cellulose, nitrocellulose, and the like. Otherexamples of insoluble polymers include, but are not limited to, lacquer,and acrylic and/or methacrylic ester polymers, polymers or copolymers ofacrylate or methacrylate having a low quaternary ammonium content, ormixture thereof and the like. Other examples of insoluble polymersinclude EUDRAGIT RS®, EUDRAGIT RL®, and EUDRAGIT NE®. Insoluble polymersuseful in the present invention include polyvinyl esters, polyvinylacetals, polyacrylic acid esters, butadiene styrene copolymers, and thelike. In one embodiment, colonic delivery is achieved by use of aslowly-eroding wax plug (e.g., various PEGS, including for example,PEG6000).

In a further embodiment, the delayed-release coating may be degraded bya microbial enzyme present in the gut flora. In one embodiment, thedelayed-release coating may be degraded by a bacteria present in thesmall intestine. In another embodiment, the delayed-release coating maybe degraded by a bacteria present in the large intestine.

In various embodiments, the modified release formulation is designed forrelease in the colon. Various colon-specific delivery approaches may beutilized. For example, the modified release formulation may beformulated using a colon-specific drug delivery system (CODES) asdescribed for example, in Li et al, AAPS PharmSciTech (2002), 3(4): 1-9,the entire contents of which are incorporated herein by reference. Drugrelease in such a system is triggered by colonic microflora coupled withpH-sensitive polymer coatings. For example, the formulation may bedesigned as a core tablet with three layers of polymer. The firstcoating is an acid-soluble polymer (e.g., EUDRAGIT E), the outer coatingis enteric, along with a hydroxypropyl methylcellulose barrier layerinterposed in between. In another embodiment, colon delivery may beachieved by formulating the alkaline phosphatase (and/or additionaltherapeutic agent) with specific polymers that degrade in the colon suchas, for example, pectin. The pectin may be further gelled or crosslinkedwith a cation such as a zinc cation. In an embodiment, the formulationis in the form of ionically crosslinked pectin beads which are furthercoated with a polymer (e.g., EUDRAGIT polymer). Additional colonspecific formulations include, but are not limited to,pressure-controlled drug delivery systems (prepared with, for example,ethylcellulose) and osmotic controlled drug delivery systems (i.e.,ORDS-CT).

Formulations for colon specific delivery of the AP-based agent (and/oradditional therapeutic agents), as described herein, may be evaluatedusing, for example, in vitro dissolution tests. For example, paralleldissolution studies in different buffers may be undertaken tocharacterize the behavior of the formulations at different pH levels.Alternatively, in vitro enzymatic tests may be carried out. For example,the formulations may be incubated in fermenters containing suitablemedium for bacteria, and the amount of drug released at different timeintervals is determined. Drug release studies can also be done in buffermedium containing enzymes or rat or guinea pig or rabbit cecal contentsand the amount of drug released in a particular time is determined. In afurther embodiment, in vivo evaluations may be carried out using animalmodels such as dogs, guinea pigs, rats, and pigs. Further, clinicalevaluation of colon specific drug delivery formulations may be evaluatedby calculating drug delivery index (DDI) which considers the relativeratio of RCE (relative colonic tissue exposure to the drug) to RSC(relative amount of drug in blood i.e. that is relative systemicexposure to the drug). Higher drug DDI indicates better colon drugdelivery. Absorption of drugs from the colon may be monitored bycolonoscopy and intubation.

In various embodiments, the present formulation provide for substantialuniform dissolution of the AP-based agent (and/or additional therapeuticagent) in the area of release in the GI tract. In an embodiment, thepresent formulation minimizes patchy or heterogeneous release of theAP-based agent.

In various embodiments, the present invention provides formodified-release formulations that release multiple doses of theAP-based agent, at different locations along the intestines, atdifferent times, and/or at different pH. In an illustrative embodiment,the modified-release formulation comprises a first dose of the AP-basedagent and a second dose of the AP-based agent, wherein the first doseand the second dose are released at different locations along theintestines, at different times, and/or at different pH. For example, thefirst dose is released at the duodenum, and the second dose is releasedat the ileum. In another example, the first dose is released at thejejunum, and the second dose is released at the ileum. In otherembodiments, the first dose is released at a location along the smallintestine (e.g., the duodenum), while the second dose is released alongthe large intestine (e.g., the ascending colon). In various embodiments,the modified-release formulation may release at least one dose, at leasttwo doses, at least three doses, at least four doses, at least fivedoses, at least six doses, at least seven doses, or at least eight dosesof the AP-based agent at different locations along the intestines, atdifferent times, and/or at different pH. Further the dual pulsedescription herein applies to modified-release formulations that releasethe AP-based agent and an additional therapeutic agent.

In various embodiments, the invention provides a formulation comprising:a core particle having a base coat comprising one or more AP-basedagents, and a delayed-release coating disposed over the coated coreparticle. The delayed-release coating may be substantially stable inacidic environments and/or gastric fluid, and/or substantially unstablein near neutral to alkaline environments or intestinal fluid therebyexposing the coated core particle to intestinal fluid. The base coatcomprising one or more AP-based agents may further comprise one or moreadditional therapeutic agents. Optionally a plurality of base coats maybe applied to the core particle each of which may contain an AP-basedagent and/or an additional therapeutic agent. In an embodiment, the coreparticle includes sucrose. In an embodiment, an AP-based agent can besprayed onto an inert core (e.g., a sucrose core) and spray-dried withan enteric layer (e.g., EUDRAGIT L30 D-55) to form pellets or beadscontaining AP-based agents.

Optionally, the core particle may comprise one or more AP-based agentsand/or one or more additional therapeutic agents. In one embodiment, oneor more doses of the AP-based agent may be encapsulated in a coreparticle, for example, in the form of a microsphere or a mini-sphere.For example, the AP-based agent may be combined with a polymer (e.g.,latex), and then formed into a particulate, micro-encapsulated enzymepreparation, without using a sucrose core. The microspheres ormini-spheres thus formed may be optionally covered with adelayed-release coating.

A variety of approaches for generating particulates (such asmicrospheres, mini-spheres, aggregates, other) may be utilized for theinclusion of enzymatic proteins. They typically involve at least twophases, one containing the protein, and one containing a polymer thatforms the backbone of the particulate. Most common are coacervation,where the polymer is made to separate from its solvent phase by additionof a third component, or multiple phase emulsions, such as water in oilin water (w/o/w) emulsion where the inner water phase contains theprotein, the intermediate organic phase contains the polymer, and theexternal water phase stabilizers that support the w/o/w double emulsionuntil the solvents can be removed to form, for example, microspheres ormini-spheres. Alternatively, the alkaline phosphatase and stabilizingexcipients (for example, trehalose, mannitol, Tween 80, polyvinylalcohol) are combined and sprayed from aqueous solution and collected.The particles are then suspended in a dry, water immiscible organicsolvent containing polymer and release modifying compounds, and thesuspension sonicated to disperse the particles.

An additional approach uses aqueous phases but no organic solvent.Specifically, the enzymatic protein, buffer components, a polymer latex,and stabilizing and release-modifying excipients are dissolved/dispersedin water. The aqueous dispersion is spray-dried, leading to coalescenceof the latex, and incorporation of the protein and excipients inparticles of the coalesced latex. When the release modifiers areinsoluble at acidic conditions but soluble at higher pHs (such ascarboxylic acid) then release from the matrix is inhibited in thegastric environment. In an embodiment, alkaline phosphatase may beinitially solubilized as an emulsion, microemulsion, or suspension andthen formulated into solid mini-spheres or microspheres. The formulationmay then be coated with, for example, a delayed-release,sustained-release, or controlled-release coating to achieve delivery ata specific location such as, for example, the intestines.

In various embodiments, the formulation may comprise a plurality ofmodified-release particles or beads or pellets or microspheres. In anembodiment, the formulation is in the form of capsules comprisingmultiple beads. In another embodiment, the formulation is in the form ofcapsules comprising multiple pellets. In another embodiment, theformulation is in the form of capsules comprising multiple microspheresor mini-spheres.

In some embodiments, before applying the delayed-release coating to thecoated core particle, the particle can optionally be covered with one ormore separating layers comprising pharmaceutical excipients includingalkaline compounds such as for instance pH-buffering compounds. Theseparating layer essentially separates the coated core particle from thedelayed-release coating.

The separating layer can be applied to the coated core particle bycoating or layering procedures typically used with coating equipmentsuch as a coating pan, coating granulator or in a fluidized bedapparatus using water and/or organic solvents for the coating process.As an alternative the separating layer can be applied to the corematerial by using a powder coating technique. The materials forseparating layers are pharmaceutically acceptable compounds such as, forinstance, sugar, polyethylene glycol, polyvinylpyrrolidone, polyvinylalcohol, polyvinyl acetate, hydroxypropyl cellulose, methyl-cellulose,ethylcellulose, hydroxypropyl methylcellulose, carboxymethylcellulosesodium and others, used alone or in mixtures. Additives such asplasticizers, colorants, pigments, fillers, anti-tacking and anti-staticagents, such as for instance magnesium stearate, sodium stearylfumarate, titanium dioxide, talc and other additives can also beincluded in the separating layer.

In some embodiments, the coated particles with the delayed-releasecoating may be further covered with an overcoat layer. The overcoatlayer can be applied as described for the other coating compositions.The overcoat materials are pharmaceutically acceptable compounds such assugar, polyethylene glycol, polyvinylpyrrolidone, polyvinyl alcohol,polyvinyl acetate, hydroxypropyl cellulose, methylcellulose,ethylcellulose, hydroxypropyl methylcellulose, carboxymethylcellulosesodium and others, used alone or in mixtures. The overcoat materials canprevent potential agglomeration of particles coated with thedelayed-release coating, protect the delayed-release coating fromcracking during the compaction process or enhance the tableting process.

In various embodiments, the formulations of the present invention takethe form of those as described in International Patent Application No.PCT/US15/54606, the entire contents of all of which are incorporatedherein by reference.

In various embodiments, the formulations of the present invention takethe form of those as described in one or more of U.S. Pat. Nos.8,535,713 and 8,9117,77 and US Patent Publication Nos. 20120141585,20120141531, 2006/001896, 2007/0292523, 2008/0020018, 2008/0113031,2010/0203120, 2010/0255087, 2010/0297221, 2011/0052645, 2013/0243873,2013/0330411, 2014/0017313, and 2014/0234418, the contents of which arehereby incorporated by reference in their entirety.

In various embodiments, the formulations of the present invention takethe form of those as described in International Patent Publication No.WO 2008/135090, the contents of which are hereby incorporated byreference in their entirety.

In various embodiments, the formulations of the present invention takethe form of those described in one or more of U.S. Pat. Nos. 4,196,564;4,196,565; 4,247,006; 4,250,997; 4,268,265; 5,317,849; 6,572,892;7,712,634; 8,074,835; 8,398,912; 8,440,224; 8,557,294; 8,646,591;8,739,812; 8,810,259; 8,852,631; and 8,911,788 and US Patent PublicationNos. 2014/0302132; 2014/0227357; 20140088202; 20130287842; 2013/0295188;2013/0307962; and 20130184290, the contents of which are herebyincorporated by reference in their entirety.

In various embodiments, the process of formulating the AP-based agent issufficiently gentle such that the tertiary structure of the AP-basedagent (e.g., dimeric structure) is substantially intact. In variousembodiments, the process of formulating the AP-based agent includes astep of refolding the AP-based agent. In such embodiments, the step ofrefolding the AP-based agent may include the addition of magnesiumand/or cyclodextrin.

Administration and Dosages

It will be appreciated that the actual dose of the AP-based agent to beadministered according to the present invention will vary according tothe particular compound, the particular dosage form, and the mode ofadministration. Many factors that may modify the action of the AP-basedagent (e.g., body weight, gender, diet, time of administration, route ofadministration, rate of excretion, condition of the subject, drugcombinations, genetic disposition and reaction sensitivities) can betaken into account by those skilled in the art. Administration can becarried out continuously or in one or more discrete doses within themaximum tolerated dose. Optimal administration rates for a given set ofconditions can be ascertained by those skilled in the art usingconventional dosage administration tests.

Individual doses of the AP-based agent can be administered in unitdosage forms (e.g., tablets or capsules) containing, for example, fromabout 0.01 mg to about 1,000 mg, about 0.01 mg to about 900 mg, about0.01 mg to about 800 mg, about 0.01 mg to about 700 mg, about 0.01 mg toabout 600 mg, about 0.01 mg to about 500 mg, about 0.01 mg to about 400mg, about 0.01 mg to about 300 mg, about 0.01 mg to about 200 mg, fromabout 0.1 mg to about 100 mg, from about 0.1 mg to about 90 mg, fromabout 0.1 mg to about 80 mg, from about 0.1 mg to about 70 mg, fromabout 0.1 mg to about 60 mg, from about 0.1 mg to about 50 mg, fromabout 0.1 mg to about 40 mg, from about 0.1 mg to about 30 mg, fromabout 0.1 mg to about 20 mg, from about 0.1 mg to about 10 mg, fromabout 0.1 mg to about 5 mg, from about 0.1 mg to about 3 mg, or fromabout 0.1 mg to about 1 mg active ingredient per unit dosage for. Forexample, a unit dosage form can be about 0.01 mg, about 0.02 mg, about0.03 mg, about 0.04 mg, about 0.05 mg, about 0.06 mg, about 0.07 mg,about 0.08 mg, about 0.09 mg, about 0.1 mg, about 0.2 mg, about 0.3 mg,about 0.4 mg, about 0.5 mg, about 0.6 mg, about 0.7 mg, about 0.8 mg,about 0.9 mg, about 1 mg, about 2 mg, about 3 mg, about 4 mg, about 5mg, about 6 mg, about 7 mg, about 8 mg, about 9 mg, about 10 mg, about11 mg, about 12 mg, about 13 mg, about 14 mg, about 15 mg, about 16 mg,about 17 mg, about 18 mg, about 19 mg, about 20 mg, about 21 mg, about22 mg, about 23 mg, about 24 mg, about 25 mg, about 26 mg, about 27 mg,about 28 mg, about 29 mg, about 30 mg, about 31 mg, about 32 mg, about33 mg, about 34 mg, about 35 mg, about 36 mg, about 37 mg, about 38 mg,about 39 mg, about 40 mg, about 41 mg, about 42 mg, about 43 mg, about44 mg, about 45 mg, about 46 mg, about 47 mg, about 48 mg, about 49 mg,about 50 mg, about 51 mg, about 52 mg, about 53 mg, about 54 mg, about55 mg, about 56 mg, about 57 mg, about 58 mg, about 59 mg, about 60 mg,about 61 mg, about 62 mg, about 63 mg, about 64 mg, about 65 mg, about66 mg, about 67 mg, about 68 mg, about 69 mg, about 70 mg, about 71 mg,about 72 mg, about 73 mg, about 74 mg, about 75 mg, about 76 mg, about77 mg, about 78 mg, about 79 mg, about 80 mg, about 81 mg, about 82 mg,about 83 mg, about 84 mg, about 85 mg, about 86 mg, about 87 mg, about88 mg, about 89 mg, about 90 mg, about 91 mg, about 92 mg, about 93 mg,about 94 mg, about 95 mg, about 96 mg, about 97 mg, about 98 mg, about99 mg, about 100 mg, about 200 mg, about 300 mg, about 400 mg, about 500mg, about 600 mg, about 700 mg, about 800 mg, about 900 mg, or about1,000 mg of the AP-based agent, inclusive of all values and rangestherebetween.

In one embodiment, the AP-based agent is administered at an amount offrom about 0.01 mg to about 1,000 mg daily, about 0.01 mg to about 900mg daily, about 0.01 mg to about 800 mg daily, about 0.01 mg to about700 mg daily, about 0.01 mg to about 600 mg daily, about 0.01 mg toabout 500 mg daily, about 0.01 mg to about 400 mg daily, about 0.01 mgto about 300 mg daily, about 0.01 mg to about 200 mg daily, about 0.01mg to about 100 mg daily, an amount of from about 0.1 mg to about 100 mgdaily, from about 0.1 mg to about 95 mg daily, from about 0.1 mg toabout 90 mg daily, from about 0.1 mg to about 85 mg daily, from about0.1 mg to about 80 mg daily, from about 0.1 mg to about 75 mg daily,from about 0.1 mg to about 70 mg daily, from about 0.1 mg to about 65 mgdaily, from about 0.1 mg to about 60 mg daily, from about 0.1 mg toabout 55 mg daily, from about 0.1 mg to about 50 mg daily, from about0.1 mg to about 45 mg daily, from about 0.1 mg to about 40 mg daily,from about 0.1 mg to about 35 mg daily, from about 0.1 mg to about 30 mgdaily, from about 0.1 mg to about 25 mg daily, from about 0.1 mg toabout 20 mg daily, from about 0.1 mg to about 15 mg daily, from about0.1 mg to about 10 mg daily, from about 0.1 mg to about 5 mg daily, fromabout 0.1 mg to about 3 mg daily, from about 0.1 mg to about 1 mg daily,or from about 5 mg to about 80 mg daily. In various embodiments, theAP-based agent is administered at a daily dose of about 0.01 mg, about0.02 mg, about 0.03 mg, about 0.04 mg, about 0.05 mg, about 0.06 mg,about 0.07 mg, about 0.08 mg, about 0.09 mg, about 0.1 mg, about 0.2 mg,about 0.3 mg, about 0.4 mg, about 0.5 mg, about 0.6 mg, about 0.7 mg,about 0.8 mg, about 0.9 mg, about 1 mg, about 2 mg, about 3 mg, about 4mg, about 5 mg, about 6 mg, about 7 mg, about 8 mg, about 9 mg, about 10mg, about 11 mg, about 12 mg, about 13 mg, about 14 mg, about 15 mg,about 16 mg, about 17 mg, about 18 mg, about 19 mg, about 20 mg, about21 mg, about 22 mg, about 23 mg, about 24 mg, about 25 mg, about 26 mg,about 27 mg, about 28 mg, about 29 mg, about 30 mg, about 31 mg, about32 mg, about 33 mg, about 34 mg, about 35 mg, about 36 mg, about 37 mg,about 38 mg, about 39 mg, about 40 mg, about 41 mg, about 42 mg, about43 mg, about 44 mg, about 45 mg, about 46 mg, about 47 mg, about 48 mg,about 49 mg, about 50 mg, about 51 mg, about 52 mg, about 53 mg, about54 mg, about 55 mg, about 56 mg, about 57 mg, about 58 mg, about 59 mg,about 60 mg, about 61 mg, about 62 mg, about 63 mg, about 64 mg, about65 mg, about 66 mg, about 67 mg, about 68 mg, about 69 mg, about 70 mg,about 71 mg, about 72 mg, about 73 mg, about 74 mg, about 75 mg, about76 mg, about 77 mg, about 78 mg, about 79 mg, about 80 mg, about 81 mg,about 82 mg, about 83 mg, about 84 mg, about 85 mg, about 86 mg, about87 mg, about 88 mg, about 89 mg, about 90 mg, about 91 mg, about 92 mg,about 93 mg, about 94 mg, about 95 mg, about 96 mg, about 97 mg, about98 mg, about 99 mg, about 100 mg, about 200 mg, about 300 mg, about 400mg, about 500 mg, about 600 mg, about 700 mg, about 800 mg, about 900mg, or about 1,000 mg, inclusive of all values and ranges therebetween.

In some embodiments, a suitable dosage of the AP-based agent is in arange of about 0.01 mg/kg to about 100 mg/kg of body weight of thesubject, about 0.01 mg/kg to about 90 mg/kg of body weight of thesubject, about 0.01 mg/kg to about 80 mg/kg of body weight of thesubject, about 0.01 mg/kg to about 70 mg/kg of body weight of thesubject, about 0.01 mg/kg to about 60 mg/kg of body weight of thesubject, about 0.01 mg/kg to about 50 mg/kg of body weight of thesubject, about 0.01 mg/kg to about 40 mg/kg of body weight of thesubject, about 0.01 mg/kg to about 30 mg/kg of body weight of thesubject, about 0.01 mg/kg to about 20 mg/kg of body weight of thesubject, about 0.01 mg/kg to about 10 mg/kg of body weight of thesubject, for example, about 0.01 mg/kg, about 0.02 mg/kg, about 0.03mg/kg, about 0.04 mg/kg, about 0.05 mg/kg, about 0.06 mg/kg, about 0.07mg/kg, about 0.08 mg/kg, about 0.09 mg/kg, about 0.1 mg/kg, about 0.2mg/kg, about 0.3 mg/kg, about 0.4 mg/kg, about 0.5 mg/kg, about 0.6mg/kg, about 0.7 mg/kg, about 0.8 mg/kg, about 0.9 mg/kg, about 1 mg/kg,about 1.1 mg/kg, about 1.2 mg/kg, about 1.3 mg/kg, about 1.4 mg/kg,about 1.5 mg/kg, about 1.6 mg/kg, about 1.7 mg/kg, about 1.8 mg/kg, 1.9mg/kg, about 2 mg/kg, about 3 mg/kg, about 4 mg/kg, about 5 mg/kg, about6 mg/kg, about 7 mg/kg, about 8 mg/kg, about 9 mg/kg, about 10 mg/kgbody weight, about 20 mg/kg body weight, about 30 mg/kg body weight,about 40 mg/kg body weight, about 50 mg/kg body weight, about 60 mg/kgbody weight, about 70 mg/kg body weight, about 80 mg/kg body weight,about 90 mg/kg body weight, or about 100 mg/kg body weight, inclusive ofall values and ranges therebetween. In other embodiments, a suitabledosage of the AP-based agent is in a range of about 0.01 mg/kg to about10 mg/kg of body weight, in a range of about 0.01 mg/kg to about 9 mg/kgof body weight, in a range of about 0.01 mg/kg to about 8 mg/kg of bodyweight, in a range of about 0.01 mg/kg to about 7 mg/kg of body weight,in a range of 0.01 mg/kg to about 6 mg/kg of body weight, in a range ofabout 0.05 mg/kg to about 5 mg/kg of body weight, in a range of about0.05 mg/kg to about 4 mg/kg of body weight, in a range of about 0.05mg/kg to about 3 mg/kg of body weight, in a range of about 0.05 mg/kg toabout 2 mg/kg of body weight, in a range of about 0.05 mg/kg to about1.5 mg/kg of body weight, or in a range of about 0.05 mg/kg to about 1mg/kg of body weight.

In accordance with certain embodiments of the invention, the AP-basedagent may be administered, for example, more than once daily (e.g.,about two, about three, about four, about five, about six, about seven,about eight, about nine, or about ten times per day), about once perday, about every other day, about every third day, about once a week,about once every two weeks, about once every month, about once every twomonths, about once every three months, about once every six months, orabout once every year.

Methods of Treatment

In some aspects, the present invention provides methods for thetherapeutic use of an AP-based agent. In an embodiment, the presentinvention provides methods for the treatment or prevention of one ormore neurodevelopmental disorders.

In various embodiments, the present methods reduce or prevent animpairment of the growth and development of the brain or central nervoussystem (CNS).

In various embodiments, the subject is a pregnant woman. In variousembodiments, the pregnant woman is afflicted with one or more ofgastrointestinal dysbiosis, obesity, metabolic syndrome, gut-mediatedsystemic inflammation, and leaky gut. In various embodiments, theoffspring of the pregnant woman is prevented from developing aneurodevelopmental disorder.

In various embodiments, the neurodevelopmental disorder is one or moreof autism spectrum disorder (ASD), schizophrenia, attention deficithyperactivity disorder (ADHD), schizoaffective disorder, and bipolaraffective disorder.

In various embodiments, the neurodevelopmental disorder is ASD.

In some aspects, the present invention provides a method of treatingautism spectrum disorder (ASD), comprising administering an effectiveamount of an AP-based agent described herein, including withoutlimitation, orally administered IAP, to a patient in need thereof.

In various embodiments, the method provides administering an AP-basedagent, including without limitation orally administered IAP, to apregnant woman afflicted with a risk factor for ASD (e.g., withoutlimitation, one or more of gastrointestinal dysbiosis, obesity,metabolic syndrome, gut-mediated systemic inflammation, and leaky gut),to reduce the likelihood of the subject's offspring from developing ASD(and/or reducing or eliminating one or more symptoms of ASD in thesubject's offspring).

ASD are a group of diseases characterized by varying degrees ofimpairment in communication skills, social interactions, and restricted,repetitive and stereotyped patterns of behavior. The difference in thediseases depends on the time of onset, the rate of symptom development,the severity of symptoms, and the exact nature of the symptoms.

These disorders range from mild to severe impairment and include suchdiseases as autism, Asperger's syndrome, PDD-NOS, Rett's disorder,childhood disintegrative disorder, semantic communication disorder,non-verbal learning disabilities, high functioning autism, hyperlexiaand some aspects of attention deficit hyperactivity disorder.

In various embodiments, the method reduces one or more symptoms of ASDas noted in the DSM-IV or other such autism-specific diagnosticmethodology. According to the Autism Society of America (ASA), autism isgenerally characterized as one of five disorders coming under theumbrella of Pervasive Developmental Disorders (PDD), a category ofneurological disorders characterized by severe and pervasive impairmentin several areas of development, including social interaction andcommunications skills (DSM-IV-TR). The five disorders under PDD, whichare treated or prevented in various embodiments of the present methodsare: autistic disorder, Asperger's Disorder, childhood disintegrativedisorder (CDD, or Heller's syndrome), Rett's Disorder or Rett'sSyndrome, and pervasive developmental disorder-not otherwise specified(PDD-NOS, or atypical autism).

Specific or Explicit diagnostic criteria for each of these disorders canbe found in the Diagnostic & Statistical Manual of Mental Disorders (DSMIV-TR) as distributed by the American Psychiatric Association (APA).

In various embodiments, the method provides treatment that is manifestedin a reversal of one or more of the DSM-IV's twelve diagnostic criteria,which fall into three categories: (1) impairments in social interaction(e.g. marked impairment in the use of multiple nonverbal behaviors suchas eye-to-eye gaze, facial expression, body postures, and gestures toregulate social interaction; failure to develop peer relationshipsappropriate to developmental level; a lack of spontaneous seeking toshare enjoyment, interests, or achievement with other people (e.g., by alack of showing, bringing, or pointing out objects of interest); andlack of social or emotional reciprocity); (2) impairments incommunication (e.g., delay in, or total lack of, the development ofspoken language (not accompanied by an attempt to compensate throughalternative modes of communication such as gesture or mime); inindividuals with adequate speech, marked impairment in the ability toinitiate or sustain a conversation with others; stereotyped andrepetitive use of language or idiosyncratic language; and lack ofvaried, spontaneous make-believe play or social imitative playappropriate to developmental level), and (3) a restricted repertoire ofactivities and interests (e.g., encompassing preoccupation with one ormore stereotyped and restricted patterns of interest that is abnormaleither in intensity or focus; apparently inflexible adherence tospecific, nonfunctional routines or rituals; stereotyped and repetitivemotor mannerisms (e.g., hand or finger flapping or twisting, or complexwhole-body movements); and persistent preoccupation with parts ofobjects).

The following traits, as identified by the ASA, may also be present inpersons with autism and are reduced or eliminated by the present methodsin various embodiments: insistence on sameness or resistance to change;difficulty in expressing needs; (i.e. uses gestures or pointing insteadof words); repeating words or phrases in place of normal, responsivelanguage; laughing, crying, showing distress for reasons not apparent toothers; prefers to be alone or aloof manner; tantrums; difficulty inmixing with others; may not want to cuddle or be cuddled; minor or noeye contact; unresponsive to normal teaching methods; sustained oddplay; spins objects; inappropriate attachments to objects; apparentover-sensitivity or under-sensitivity to pain; no real fears of danger;noticeable physical over-activity or extreme under-activity; and unevengross/fine motor skills; and/or not responsive to verbal cues (i.e. actsas if deaf although hearing tests in normal range).

In various embodiments, the present methods may be useful in treatingone or more symptoms or characteristics of ASD, which include, by way ofnon-limiting example, stereotyped movements, social withdrawal andaverted gaze including an inability to make eye contact, repetitivebehaviors and obsessions, anxiety, attention deficit, hyperactivity,depression, a reclusive personality, and the inability to understandfeelings. Patients afflicted with ASD may have an aversion to physicalaffection or contact, ignore communication from others, or if sociallyengaged, demonstrate a marked inability to communicate or relate toothers. Communication difficulties may manifest as a monotone voice, aninability to control the volume of their voice, echolalia or aninability to talk at all. Individuals with autism spectrum disorders mayalso suffer from visual difficulties, comprehension difficulties, soundand light sensitivity and mental retardation.

In various embodiments, the present methods may be useful in treatingone or more symptoms or characteristics of ASD, which include, by way ofnon-limiting example, reduced communication message skills (e.g. notspeaking or very limited speech; loss of words the child was previouslyable to say; difficulty expressing basic wants and needs; poorvocabulary development; problems following directions or finding objectsthat are named; repeating what is said (echolalia); problems answeringquestions; and speech that sounds different (e.g., “robotic” speech orspeech that is high-pitched) and or reduced social community skills(e.g. poor eye contact with people or objects, poor play skills (pretendor social play), being overly focused on a topic or objects thatinterest them, problems making friends, crying, becoming angry,giggling, or laughing for no known reason or at the wrong time, anddisliking being touched or held); various reduced response mechanisms(e.g. rocking, hand flapping or other movements (self-stimulatingmovements), not paying attention to things the child sees or hears,problems dealing with changes in routine, using objects in unusual ways,unusual attachments to objects, no fear of real dangers, being eithervery sensitive or not sensitive enough to touch, light, or sounds (e.g.,disliking loud sounds or only responding when sounds are very loud; alsocalled a sensory integration disorder), feeding difficulties (acceptingonly select foods, refusing certain food textures), and sleep problems).

The effectiveness of the AP-based agents for these and relatedconditions can be demonstrated according to a variety of methods,including, for example, by measuring markers such as those measured inthe Checklist of Autism in Toddlers (CHAT), the modified Checklist forAutism in Toddlers (M-CHAT), the Screening Tool for Autism inTwo-Year-Olds (STAT), the Social Communication Questionnaire (SCQ), theAutism Spectrum Screening Questionnaire (ASSQ), the Australian Scale forAsperger's Syndrome, the Childhood Asperger Syndrome Test (CAST), theAutism Diagnosis Interview-Revised (ADI-R), the Autism DiagnosticObservation Schedule (ADOS-G), the Childhood Autism Rating Scale (CARS),audiologic hearing evaluation, Administered PTSD Scale, the EysenckPersonality Inventory, the Hamilton Anxiety Scale, or in various animalmodels such as the well-known Vogel (thirsty rat conflict) test, or theelevated plus maze test. Effective amounts of the present compounds andcompositions (and, optionally, an additional therapeutic agent) willmeasurably prevent, decrease the severity of, or delay the onset orduration of, one or more of the foregoing autism spectrum disorders orrelated disorders of symptoms of such disorders in a patient. Further,the DSM-5, e.g. the section entitled “ASD and Social CommunicationDisorder,” which is hereby incorporated by reference in its entirety,and the International Statistical Classification of Diseases and RelatedHealth Problems-10th Revision (ICD-10) can be used as diagnosticclassifications for ASD. In some embodiments, stereotypy is useful as adiagnostic for ASD, including in children with autism.

In some embodiments, the terms “patient” and “subject” are usedinterchangeably. In some embodiments, the subject and/or animal is amammal, e.g., a human, mouse, rat, guinea pig, dog, cat, horse, cow,pig, rabbit, sheep, or non-human primate, such as a monkey, chimpanzee,or baboon. In other embodiments, the subject and/or animal is anon-mammal, such, for example, a zebrafish.

In various embodiments, methods of the invention are useful in treatinga human subject. In some embodiments, the human is a pediatric human. Inother embodiments, the human is an adult human. In other embodiments,the human is a geriatric human. In other embodiments, the human may bereferred to as a patient. In some embodiments, the human is a female. Insome embodiments, the human is a male.

As described elsewhere herein, in various embodiments, the human subjectis a pregnant female. As described elsewhere herein, in variousembodiments, the human subject is an unborn child.

In certain embodiments, the human has an age in a range of from about 1to about 18 months old, from about 18 to about 36 months old, from about1 to about 5 years old, from about 5 to about 10 years old, from about10 to about 15 years old, from about 15 to about 20 years old, fromabout 20 to about 25 years old, from about 25 to about 30 years old,from about 30 to about 35 years old, from about 35 to about 40 yearsold, from about 40 to about 45 years old, from about 45 to about 50years old, from about 50 to about 55 years old, from about 55 to about60 years old, from about 60 to about 65 years old, from about 65 toabout 70 years old, from about 70 to about 75 years old, from about 75to about 80 years old, from about 80 to about 85 years old, from about85 to about 90 years old, from about 90 to about 95 years old or fromabout 95 to about 100 years old.

Additional Therapeutic Agents and Combination Therapy

Administration of the present compositions and formulations comprisingthe AP-based agent may be combined with additional therapeutic agents.Co-administration of the additional therapeutic agent and the presentcompositions/formulations may be simultaneous or sequential. Further,the present compositions/formulations may comprise an additionaltherapeutic agent (e.g. via co-formulation). For example, the additionaltherapeutic agent and the AP-based agent may be combined into a singleformulation. Alternatively, the additional therapeutic agent and theAP-based agent may be formulated separately.

In one embodiment, the additional therapeutic agent and the AP-basedagent are administered to a subject simultaneously. The term“simultaneously” as used herein, means that the additional therapeuticagent and the AP-based agent are administered with a time separation ofno more than about 60 minutes, such as no more than about 30 minutes, nomore than about 20 minutes, no more than about 10 minutes, no more thanabout 5 minutes, or no more than about 1 minute. Administration of theadditional therapeutic agent and the AP-based agent can be bysimultaneous administration of a single formulation (e.g., a formulationcomprising the additional therapeutic agent and the alkalinephosphatase) or of separate formulations (e.g., a first formulationincluding the additional therapeutic agent and a second formulationincluding the AP-based agent).

In a further embodiment, the additional therapeutic agent and theAP-based agent are administered to a subject simultaneously but therelease of the additional therapeutic agent and the alkaline phosphatasefrom their respective dosage forms (or single unit dosage form ifco-formulated) may occur sequentially.

Co-administration does not require the additional therapeutic agent andthe AP-based agent to be administered simultaneously, if the timing oftheir administration is such that the pharmacological activities of theadditional therapeutic agent and the AP-based agent overlap in time. Forexample, the additional therapeutic agent and the AP-based agent can beadministered sequentially. The term “sequentially” as used herein meansthat the additional therapeutic agent and the AP-based agent areadministered with a time separation of more than about 60 minutes. Forexample, the time between the sequential administration of theadditional therapeutic agent and the AP-based agent can be more thanabout 60 minutes, more than about 2 hours, more than about 5 hours, morethan about 10 hours, more than about 1 day, more than about 2 days, morethan about 3 days, or more than about 1 week apart. The optimaladministration times will depend on the rates of metabolism, excretion,and/or the pharmacodynamic activity of the additional therapeutic agentand the AP-based agent being administered. Either the additionaltherapeutic agent or the AP-based agent may be administered first.

Co-administration also does not require the additional therapeutic agentand the AP-based agent to be administered to the subject by the sameroute of administration. Rather, each therapeutic agent can beadministered by any appropriate route, for example, parenterally ornon-parenterally.

In various embodiments, the present agents are used in conjunction withapplied behavior analysis or other behavior modification techniques;dietary alteration such as a gluten or casein free diet; vitamin B6,optionally combined with magnesium; and one or more additional agents.

In various embodiments, the additional agents are neurotransmitterreuptake inhibitors (e.g. fluoxetine), tricyclic antidepressants (e.g.imipramine), anticonvulsants (e.g. lamotrigine), atypical antipsychotics(e.g. clozapine), acetylcholinesterase inhibitors (e.g. rivastigmine).

In various embodiments, the additional agent is an anti-anxiety and/oranti-depression agent such as fiuoxetine, fiuvoxamine, sertraline andclomipramine. In various embodiments, the additional agent is anantipsychotic medication such as chlorpromazine, thioridazine, andhaloperidol. In various embodiments, the additional agent is ananticonvulsant agent such as arbamazepine, lamotrigine, topiramate, andvalproic acid.

EXAMPLES Example 1 Stability of AP-Based Agent in Chyme

The stability of various AP-based agents in chyme is assessed. Chymespecimens (5 individual and 1 mixed) are first evaluated for backgroundalkaline phosphatase activity prior to use in analysis, and chymespecimens with the lowest amount of background activity are used for thestability study. Three separate AP proteins, hiAP, biAP, and a hiAP-FCfusion are incubated at 37° C. in a HEPES buffer containing 5% clarifiedhuman chyme. Two aliquots from each sample are removed at 0, 30, 60,120, 180, and 240 minutes of incubation. One aliquot is immediatelymixed with Laemli sample buffer for SDS-PAGE analysis and the other isimmediately mixed with a protease inhibitor cocktail and stored frozenfor analysis of AP activity. The samples are also incubated in HEPESbuffer alone and aliquots removed at 0 and 240 minutes as controls.Collected samples are subjected to SDS-PAGE and the products ofincubation examined by Coomassie blue staining.

Alkaline phosphatase activity before and after incubation in chyme isexamined using a commercial kit (Abcam). It is expected that allAP-based agents remain stable in chyme for the entire duration of theexperiment. Additionally, there is no reduction in AP activity afterchyme incubation, which confirms that the AP-based agents are notdegraded in chyme under the tested conditions.

Example 2 Engineering Bacterial AP-based Agent to Increase CatalyticActivity by Specific Amino Acids Changes

There are some functional differences between the bacterial andmammalian APs. By and large, the mammalian enzymes exhibit 20-30-foldhigher catalytic activity as well as a shift in the pH of optimalactivity towards higher pH. Some mammalian alkaline phosphatases alsorequire magnesium in order to achieve maximal activity. In addition, itis not known whether bacterial AP maintains the same de-phosphorylationpattern as the mammalian APs. By nucleotide comparison with mammalianAP, the bacterial Escherichia coli AP has been successfully engineeredto achieve activity similar to the mammalian AP. Several residues havebeen mutagenized and AP activity assessed. Previous work indicated thatthe D101S mutant in Escherichia coli AP, which contains an Asp/Serreplacement within the —Asp101-Ser102-Ala103— region of the activecenter, showed a 10-fold higher activity over the wild-type AP ((Zhang,F. Appl. Biochem. Biotechnol. 2002;101:197-210). Double mutants such asD153H/K328H resulted in enhanced activity and properties of E. coli APsimilar to the mammalian alkaline phosphatases, and the D153H/K328Hmutant enzyme is 5.6-fold more active than the wild-type enzyme.Furthermore, the double mutant D153G/D330N is as active as the mammalianAP, with 40- to 50-fold higher activity than that of the wild-typebacterial enzyme (Le Du M-H., 2002; Murphy, JE., 1994; Muller, BH.,2001).

To engineer the BSAP IV, the BSAP IV sequence disclosed herein (e.g.,SEQ ID NO:17) is synthesized de novo with single, double, triple orquadruple mutations at positions D101A, W328H, A330N and G3740. The BSAPvariants are tested in various in vitro and in vivo assays for theirtherapeutic potentials.

Example 3 Engineering Bacterial AP-Based Agent to Increase CatalyticActivity by Directed Evolution

Error-prone PCR (Leung, D. Technique, 1989;1:11-15) and DNA shufflingare utilized to identify mutations in the mammalian and bacterial APgene that can confer an increased activity.

For example, for the error-prone PCR, specific primers are used toamplify regions of the BSAP IV gene. Primers are designed to amplifyspecific regions of the BSAP IV coding sequence that do not affectmutation already known to increase BSAP IV activity. The PCR parametersare as follow: 1 mM dCTP, 1 mM dTTP, 0.2 mM dATP, 0.2 mM dGTP, 7 mMMg²⁺, 0.05 mM Mn²⁺ 50 ng of each primer, 1×Taq DNA polymerase buffer, 10ng of DNA template and 2.5 units of Taq DNA polymerase in 50 ml finalvolume. The reaction is subjected to 25 cycles as follows: 1 minute at94° C., 1 minute at 56° C., and 1.5 minutes at 72° C. to generate anerror frequency of approximately 1 to 2 substitutions per 1000 bases.The amplified products are digested appropriate restrictionendonucleases, followed by the ligation with the same digested templatevector. The E. coli SL21 (DE3) containing sequences from the error pronePCR is then transformed with the ligation mixture to create the mutantlibrary (Moore, J C. Nat. Biotechnol. 1996; 14:458-467).

DNA shuffling is performed as described by Stemmer (Proc. Natl. Acad.Sci. USA, 1994;91:10747-10751) and Lorimer and Pastan (Nucleic AcidsRes. 1995;23:3067-3068) with some modifications (Xu, HF. et al 2003). Atotal quantity of 5 mg BSAP IV fragments is randomly fragmented usingDNase I for 15 minutes. The digested DNA fragments are visualized as asmall smear on a 2% low melting temperature agarose gel. The fragmentsin specified molecular size ranges are subjected to gel extraction andthen eluted with 30 ml elution buffer (10 mM Tris, 1 mM EDTA, pH 8.0).Reassembly of the DNA fragments was conducted by PCR without primers,using the following conditions: 94° C. for 4 minute, then 40 cycles of94° C. for 50 seconds, 56° C. for 50 seconds, 72° C. for 50 seconds+5seconds/cycle, followed by a final extension step at 72° C. for 7minutes. The reassembled DNA is amplified by the following procedurewith two flanking primers, and the final PCR products are ligated intopET vector and then transformed into E. coli SL21 (DE3).

The screening of mutant libraries is carried out by assessing activityof cell-free medium. The members of the mutant libraries are allowed togrow for 16-18 hours on LB plates with ampicillin and indicatorsubstrate 5-bromo-4-chloro-3-indolyl phosphate, which can bede-phosphorylated by AP resulted in blue colonies. Each active (i.e.blue) colony is then picked and suspended in a unique well of a 96-wellplate containing 200 ml of media. The cells are then treated for growthconditions and activity assay. Clones with improved activity are thensequenced.

Example 4 In Vivo Disease Models to Assess Efficacy of AP-Based Agent inAutism Spectrum Disorders

An in vivo model was established in order to perform behavioral testingon offspring.

C57BL/6J female mice were divided into treatment groups (Treatment A, B,or C) and fed either regular chow or high fat chow for 8 weeks to induceobesity in the high fat fed group (Table 1). The high fat chow wasprovided for 8 weeks before breeding, and throughout gestation andnursing.

TABLE 1 Summary of Treatment Groups Group Feed Treatment A Normal dietvehicle B High fat diet vehicle C High fat diet IAP

From the time of high fat chow feeding, mice in the experimental groupreceived the test article Bovine Intestinal Alkaline Phosphatase II(“SYN BIAPII”) at 800 U/ml, administered in the drinking water. Controlmice received vehicle water. SYN BIAPII or vehicle administrationcontinued during breeding, throughout gestation, and until weaning.

After 8 weeks of high fat chow feeding, females were mated with normalC57BL/6J mice and impregnated. The diets and compound regimens remainedthe same until weaning. Within 48 hours of parturition, litters wereassessed and offspring gender was determined. Litters were then leftundisturbed except for normal husbandry procedures until weaning.Weaning occurred 21 days after parturition.

Preliminary results are shown in Table 2. Four dams in the control group(regular chow; Treatment Group A) gave birth to twenty-five pups, total,equally distributed by gender. Six dams that received high fat chow andvehicle (Treatment Group B) gave birth to a total of fifteen newbornpups. Interestingly, as shown in the table, fourteen of these offspringare male and only one is female. In contrast, administration of IAP tosix dams fed with the high fat chow (Treatment Group C) resulted in 8newborn pups, equally distributed by gender, suggesting that IAPadministration may play a role in restoring a gender distributionimbalance when subjects ingested a high fat diet.

TABLE 2 Preliminary result of offspring from mice fed with a high fatdiet Litter Purpose Feed Treatment ID Status of 1^(st) litter Status of2^(nd) Litter Status of 3^(rd) Litter Notes ♀ ♂ Normal Lab vehicle  1 3pups (3♀), weaned 7 Pups (3♀/4♂) — 13 12 Control Diets  2 8 pups(3♀/4♂), **Stopped breeding, — 5001 weaned have reached (Normal 12males** diet)  3 3 pups (2♀/1♂), Bred again, due — weaned week of Dec 18 4 First litter lost 5 Pups (2♀/3♂) — from PND 19-21 (had 2♀/1♂) Highfat Res Diet vehicle  5 First litter lost 3 pups (3♂), weaned —  1 14control D12492  6 First litter lost Second litter lost — (High  7 Firstlitter lost 1 pup (1♂), weaned — fat)  8 First litter lost 3 Pups (3♂) — 9 1 pup (1♂), weaned 4 Pups (1♀/3♂) — 10 Hot litter lost 3 pups (3♂),weaned — High fat + Res Diet IAP 11 First litter lost Second litter lostRe-breeding  4  4 treatment D12492 12 First litter lost 1 pup (1♂),weaned Re-breeding (High 13 First litter lost Second litter lostRe-breeding fat) 14 First litter lost Second litter lost Bred again, dueweek of Jan 8 15 4 pups (2♀/2♂), 3 Pups (2♀/1♂) weaned 16 First litterlost Re-breeding Breeding was confirmed but no litter was born.Re-breeding

Example 5 Studies Assessing Efficacy of AP-Based Agent in AutismSpectrum Disorders

The purpose of this example is to assess the efficacy of SYN BIAPII foralleviating maternal diet-induced behavioral deficits in offspring.

An in vivo model was utilized to verify the therapeutic potential ofAP-based agents in a maternal autism-like model.

Mice were bred as described in Example 4.

Sixteen female C57BL/6J mice were assigned to one of three treatmentgroups: (1) regular diet with regular water (n=4); (2) high fat diet(HFD) with regular water (n=6); and (3) high fat diet with water dosedwith 800 U/mL SYN BIAPI I (n=6). Water consumption was measured daily,and the mean water consumption over the first 8 weeks is depicted inFIG. 1. The water consumption for the 14 days surrounding parturition isdisplayed in FIG. 2. Females were weighed 24 hours after arrival, andthen once per week thereafter until parturition. FIG. 3 depicts damweights over the course of the first 8 weeks, and FIG. 4 shows damweights over the course of 32 weeks. After a minimum of 8 weeks on adiet, the female mice were fasted for about 4 hours and blood was drawnto assess metabolic syndrome onset and to record blood glucose levels inorder to determine whether the presence or absence of treatment had aneffect on blood glucose levels. FIG. 5 depicts the results of the bloodglucose testing, and as can be seen from the results, there was nostatistically significant effect seen in blood glucose concentrationsafter 8 weeks of feeding, although mice receiving a high fat diet tendedto have higher blood glucoses concentrations than mice receiving normalchow. Then the female mice were paired with male C57BL/6J mice (n=6) tobreed.

Once mating was confirmed, dams were single housed. Pregnant mice wereleft undisturbed and remained on the same diet as before until pupassessment post-parturition.

Within 3-5 days of parturition, all litters were assessed, and thenumber of males and females was determined. The pups were weaned betweenpost-natal day 20-22, at which time they were weighed weekly (resultsdepicted in FIG. 6), sorted into new cages by sex and treatment group,and ear notched for tracking purposes. All pups were provided standardrodent chow and had access to regular water.

Male offspring from the aforementioned litters of C57BL/6J mice wereutilized for these studies. There was a maximum of 12 mice per treatmentfor behavioral testing. Behavioral testing was performed on weaned maleoffspring at between postnatal 7-12 weeks of age. Up to twelve offspringper treatment underwent a series of behavioral tests to determine ifbehavioral differences exist between treatment groups. Behavioraltesting was undertaken by evaluating one or more of reciprocal socialinteraction test, three-chamber paradigm test (e.g. Crawley'ssociability and preference for social novelty protocol), marble buryingassay, and activity in an open field (e.g. locomotion). All offspringunderwent the tests in the same order and within the same postnatal weekof age.

Three-Chamber Social Interaction Test

The purpose of the three-chamber social interaction test, among otherthings, is to assess sociability and preference for novelty. Phase 1 ofthe Three-Chamber Social Interaction Test allows for baseline evaluationto determine if a bias for chamber preference is pre-existing. Phase 2presentation provides an indication of “sociability” as a normal mousewill tend to spend more time in the same room and interacting with thenovel rodent rather than the novel inanimate object. Phase 3presentation is indicative of social novelty seeking behaviors; normalrodents will typically prefer to spend more time in the same room as andinteracting with the novel rodent. Preference for social novelty alsocontains components of social recognition and social memory, so isuseful for investigation of these types of measures as well.

The Three-Chamber test was administered during postnatal weeks 8-10.This test arena consisted of three equally sized rooms (20×45 cm each),divided by clear Plexiglas, and with an access door between eachcompartment. The test occurred in three distinct stages; (1)Acclimatization phase (baseline): The test animal was placed in thecenter compartment (zone) and allowed to freely explore the entire(empty) maze for 10 minutes; (2) Sociability phase (novel mouse vs novelobject vs center): A “stranger” (an unfamiliar male mouse) was containedwithin a wire mesh container in an outer chamber of the maze and anidentical container (clean and empty) was placed in the chamber at theopposite side of the arena (position of “stranger” and “novel object”was counterbalanced between trials); and (3) Social novelty phase(familiar mouse vs novel mouse vs center): A new unknown “stranger” wasplaced into the “novel object” compartment, thus providing the testanimal with a choice between spending time with a now familiar animal,or a new (novel) animal. The test mouse was returned to its holding cagebetween each test phase (intertrial interval; 2 minutes). Phase 1 wasused as a baseline to ensure there was no potential confound frominitial preference for one chamber over the other. Measurements duringPhase 2 and 3 included number of entries and time spent in each chamberas well as number and time spent directly interacting with each mouse(familiar or stranger) or the object.

The results of the three-chamber interaction test showed that anevaluation of baseline exploration revealed no bias for left or rightchambers within any treatment groups. During the sociability phase,there were no between-group differences in time spent or number ofentries into any of the 3 chambers (stranger mouse, novel object, orcenter; p>0.05). However, all groups did show a preference forinteracting with the stranger mouse as compared to the novel objectacross a number of measures. In this study, Tx refers to the testarticle SYN BIAPII. Control+HFD mice (p=0.014, paired samples t-test)and Tx+HFD mice (p=0.042, paired samples t-test) spent significantlymore time in the chamber containing the stranger mouse vs the chambercontaining the novel object (FIG. 7). In addition, the Control+HFD group(p=0.003, paired samples t-test) and the Tx+HFD group (p=0.019, pairedsamples t-test) spent significantly more time interacting with thestranger mouse vs the novel object (FIG. 8), and Control+HFD mice alsoshowed an increased number of interactions with the stranger mouse ascompared to the novel object (p=0.045, paired samples t-test) (FIG. 9).

In the social novelty phase, the Control+HFD group spent significantlymore time in the chamber containing the new stranger mouse compared tothe chamber containing the familiar mouse (p=0.005, paired samplest-test; FIG. 10), and tended toward a greater time spent interactingwith the new stranger mouse compared to the familiar mouse (p=0.058,paired samples t-test; FIG. 11). As in the sociability phase, Tx+HFDmice appeared to perform more similarly to controls for these measures,with the exception of the number of interactions, where Tx+HFD groupdisplayed a significantly increased number of interactions with thestranger mouse compared to the familiar mouse (p=0.037, paired samplest-test; FIG. 12). All mice showed a trend toward more entries into thechamber containing the stranger mouse; this was statisticallysignificant in the Normal Control group (p=0.016, paired samples t-test;FIG. 13).

An analysis of total distance travelled throughout testing was conducted(FIG. 14). There were no significant differences between groups indistance travelled during baseline testing (Stage 1) while the arena wasempty. However, during sociability testing (Stage 2), there was astatistically significant main effect (p=0.040, one way ANOVA), with theTx+HFD group moving more than Normal Controls (p=0.033, Tukey's posthoc). In addition, a trend was also seen for this measure between Tx+HFDand Control+HFD groups (p=0.116, Tukey's post hoc). This same effect wasnoted during social novelty testing (Stage 3) as well, where a mainaffect was also found (p=0.004, one-way ANOVA). The Tx+HFD group moved astatistically significantly greater distance as compared to the NormalControls (p=0.002, Tukey's post hoc) and showed a tendency toward movinga greater distance than the Control+HFD group (p=0.078).

Overall, the results showed that the HFD-derived offspring seemed tointeract less with other mice, while the IAP-derived offspring behavedsimilarly to controls, which suggests that administration of IAP reducesthe social deficits compared to no treatment.

Reciprocal Social Interaction

The purpose of the reciprocal social interaction test is to assessoverall sociability and complex social measures.

Dyadic testing was performed during postnatal weeks 10-12. Mice weresimultaneously placed in an open, unfamiliar arena (25×25 cm) witheither a familiar cage-mate, or an unfamiliar partner (with the order oftesting counterbalanced across all groups). In all cases, pairs werefrom the same treatment group. Mice were allowed to interact freely for5 minutes. Latency to first interaction, number of and time spent inbidirectional interactions (e.g. nose-to-nose sniffing), and total timespent interacting was quantified. Interaction was defined as any of thefollowing: bidirectional encounters, close following, touching partner,allogrooming, nose-to-anus sniffing, and crawling over/under.

As depicted by FIG. 15, an analysis of the number of reciprocal socialinteractions (head-to-head interacting) revealed a significant maineffect for treatment group (p=0.011, one-way ANOVA), with post-hoctesting showing the Control+HFD group animal performing a greater numberof these interactions as compared to both the Normal Control group(p=0.022, Tukey's post hoc) and Tx+HFD group (p=0.046, Tukey's posthoc). Similarly, the number of following interactions (nose-to-tailinteracting) was also increased in this group (main effect p=0.005,one-way ANOVA) with post-hoc testing showing a difference between theControl+HFD group and Normal Controls (p=0.004, Tukey's post hoc), butnot from Tx+HFD mice (p=0.156, Tukey's post hoc).

An analysis of total time spent interacting in reciprocal behavior(head-to-head interacting) showed no between-group differences, althoughfollowing behavior (nose-to-tail interacting) did reveal a trend towardan increase in following for the Control+HFD group compared to NormalControls (p=0.058, Tukey's post hoc; FIG. 16).

As shown in FIG. 17, an analysis of the mean duration of each reciprocal(head-to-head) interaction revealed a statistically significant maineffect for treatment group (p=0.022, one-way ANOVA, Welch correction).While post hoc analyses did not show any statistically significantdifferences between groups, there was a trend towards a decreasedcontact duration for Control+HFD mice compared to both the NormalControl group (p=0.081, Games-Howell post hoc) and Tx+HFD group(p=0.075, Games-Howell post hoc). No differences were observed betweengroups for mean contact duration for following (head-to-tail). Overall,the results suggest that IAP-derived mice behave more like the controlgroup than HFD-derived mice, which exhibited a lack of interaction.

In addition to social measures during reciprocal social interactiontesting, jumping behaviors were also quantified when it was noted thatsome mice were repeatedly and persistently jumping in the test arena. Asshown in FIG. 18, a main effect for treatment group was found for totalnumber of jumps during testing with a stranger mouse (p=0.009, one-wayANOVA, Welch correction), with the Control+HFD group jumping more thanboth the Normal Control group (p=0.005, Games-Howell post-hoc) and theTx+HFD group (p=0.009, Games-Howell post hoc). This effect was alsoobserved when these mice were tested with a familiar cage mate. However,as no Tx+HFD mice jumped during this testing phase, analysis using ANOVAwas not possible. Instead, t-tests were run to look for groupdifferences, and revealed that the Control+HFD group jumped more thanboth the Normal Control group (p<0.001, independent samples t-test) andTx+HFD group (p<0.001, independent samples t-test).

Overall conclusions from these behavioral studies show that offspringfrom high fat diet (HFD) SYN BIAPII litters performed more similarly tonormal controls in reciprocal interaction testing and three-chambertests. This suggests that treatment with SYN BIAPI I appears toameliorate some of the social deficits noted in offspring as aconsequence of maternal high fat diet.

Definitions

As used herein, “a,” “an,” or “the” can mean one or more than one.

Further, the term “about” when used in connection with a referencednumeric indication means the referenced numeric indication plus or minusup to 10% of that referenced numeric indication. For example, thelanguage “about 50%” covers the range of 45% to 55%.

An “effective amount,” when used in connection with medical uses is anamount that is effective for providing a measurable treatment,prevention, or reduction in the rate of pathogenesis of a disorder ofinterest.

As used herein, something is “decreased” if a read-out of activityand/or effect is reduced by a significant amount, such as by at leastabout 10%, at least about 20%, at least about 30%, at least about 40%,at least about 50%, at least about 60%, at least about 70%, at leastabout 80%, at least about 90%, at least about 95%, at least about 97%,at least about 98%, or more, up to and including at least about 100%, inthe presence of an agent or stimulus relative to the absence of suchmodulation. As will be understood by one of ordinary skill in the art,in some embodiments, activity is decreased and some downstream read-outswill decrease but others can increase.

Conversely, activity is “increased” if a read-out of activity and/oreffect is increased by a significant amount, for example by at leastabout 10%, at least about 20%, at least about 30%, at least about 40%,at least about 50%, at least about 60%, at least about 70%, at leastabout 80%, at least about 90%, at least about 95%, at least about 97%,at least about 98%, or more, up to and including at least about 100% ormore, at least about 2-fold, at least about 3-fold, at least about4-fold, at least about 5-fold, at least about 6-fold, at least about7-fold, at least about 8-fold, at least about 9-fold, at least about10-fold, at least about 50-fold, at least about 100-fold, in thepresence of an agent or stimulus, relative to the absence of such agentor stimulus.

As referred to herein, all compositional percentages are by weight ofthe total composition, unless otherwise specified. As used herein, theword “include,” and its variants, is intended to be non-limiting, suchthat recitation of items in a list is not to the exclusion of other likeitems that may also be useful in the compositions and methods of thistechnology. Similarly, the terms “can” and “may” and their variants areintended to be non-limiting, such that recitation that an embodiment canor may comprise certain elements or features does not exclude otherembodiments of the present technology that do not contain those elementsor features.

Although the open-ended term “comprising,” as a synonym of terms such asincluding, containing, or having, is used herein to describe and claimthe invention, the present invention, or embodiments thereof, mayalternatively be described using alternative terms such as “consistingof” or “consisting essentially of.”

As used herein, the words “preferred” and “preferably” refer toembodiments of the technology that afford certain benefits, undercertain circumstances. However, other embodiments may also be preferred,under the same or other circumstances. Furthermore, the recitation ofone or more preferred embodiments does not imply that other embodimentsare not useful, and is not intended to exclude other embodiments fromthe scope of the technology.

The amount of compositions described herein needed for achieving atherapeutic effect may be determined empirically in accordance withconventional procedures for the particular purpose. Generally, foradministering therapeutic agents (e.g., additional therapeutic agentsdescribed herein) for therapeutic purposes, the therapeutic agents aregiven at a pharmacologically effective dose. A “pharmacologicallyeffective amount,” “pharmacologically effective dose,” “therapeuticallyeffective amount,” or “effective amount” refers to an amount sufficientto produce the desired physiological effect or amount capable ofachieving the desired result, particularly for treating the disorder ordisease. An effective amount as used herein would include an amountsufficient to, for example, delay the development of a symptom of thedisorder or disease, alter the course of a symptom of the disorder ordisease (a g., slow the progression of a symptom of the disease), reduceor eliminate one or more symptoms or manifestations of the disorder ordisease, and reverse a symptom of a disorder or disease. Therapeuticbenefit also includes halting or slowing the progression of theunderlying disease or disorder, regardless of whether improvement isrealized.

Effective amounts, toxicity, and therapeutic efficacy can be determinedby standard pharmaceutical procedures in cell cultures, tissue samples,tissue homogenates or experimental animals, e.g., for determining theLD50 (the dose lethal to about 50% of the population) and the ED50 (thedose therapeutically effective in about 50% of the population). Thedosage can vary depending upon the dosage form employed and the route ofadministration utilized. The dose ratio between toxic and therapeuticeffects is the therapeutic index and can be expressed as the ratioLD50/ED50. In some embodiments, compositions and methods that exhibitlarge therapeutic indices are preferred. A therapeutically effectivedose can be estimated initially from in vitro assays, including, forexample, cell culture assays or measurements or methane production instool samples. Also, a dose can be formulated in animal models toachieve a circulating plasma concentration range that includes the 1050as determined in cell culture, or in an appropriate animal model. Levelsof the described compositions in plasma can be measured, for example, byhigh performance liquid chromatography. The effects of any particulardosage can be monitored by a suitable bioassay. The dosage can bedetermined by a physician and adjusted, as necessary, to suit observedeffects of the treatment.

In certain embodiments, the effect will result in a quantifiable changeof at least about 10%, at least about 20%, at least about 30%, at leastabout 50%, at least about 70%, or at least about 90%. In someembodiments, the effect will result in a quantifiable change of about10%, about 20%, about 30%, about 50%, about 70%, or even about 90% ormore. Therapeutic benefit also includes halting or slowing theprogression of the underlying disease or disorder, regardless of whetherimprovement is realized.

As used herein, “methods of treatment” are equally applicable to use ofa composition for treating the diseases or disorders described hereinand/or compositions for use and/or uses in the manufacture of amedicaments for treating the diseases or disorders described herein.

EQUIVALENTS

While the invention has been described in connection with specificembodiments thereof, it will be understood that it is capable of furthermodifications and this application is intended to cover any variations,uses, or adaptations of the invention following, in general, theprinciples of the invention and including such departures from thepresent disclosure as come within known or customary practice within theart to which the invention pertains and as may be applied to theessential features hereinbefore set forth and as follows in the scope ofthe appended claims.

Those skilled in the art will recognize, or be able to ascertain, usingno more than routine experimentation, numerous equivalents to thespecific embodiments described specifically herein. Such equivalents areintended to be encompassed in the scope of the following claims.

INCORPORATION BY REFERENCE

All patents and publications referenced herein are hereby incorporatedby reference in their entireties.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the present invention isnot entitled to antedate such publication by virtue of prior invention.

As used herein, all headings are simply for organization and are notintended to limit the disclosure in any manner. The content of anyindividual section may be equally applicable to all sections.

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1. A method of treating or preventing a neurodevelopmental disorder in asubject in need thereof comprising, administering to the subject analkaline phosphatase (AP)-based agent.
 2. The method of claim 1, whereinthe AP-based agent is a mammalian or bacterial alkaline phosphatase. 3.The method of claim 2, wherein the bacterial AP has catalytic activitycomparable to that of mammalian AP.
 4. The method of claim 2, whereinthe mammalian alkaline phosphatase is selected from intestinal alkalinephosphatase (IAP), placental alkaline phosphatase (PLAP), germ cellalkaline phosphatase (GCAP), and tissue non-specific alkalinephosphatase (TNAP).
 5. The method of any of the above claims, whereinthe AP-based agent is intestinal alkaline phosphate (IAP).
 6. The methodof any of the above claims, wherein the neurodevelopmental disorder isselected from autism spectrum disorder (ASD), schizophrenia, attentiondeficit hyperactivity disorder (ADHD), schizoaffective disorder, andbipolar affective disorder.
 7. The method of claim 1, wherein theneurodevelopmental disorder is ASD.
 8. The method of claim 7, whereinthe treatment or prevention of ASD comprises reduction or elimination ofone or more of social withdrawal, averted gaze, inability to make eyecontact, repetitive behaviors, obsessions, anxiety, stereotypedmovements, attention deficit, hyperactivity, depression, a reclusivepersonality, and the inability to understand feelings.
 9. The method ofany of the above claims, wherein the subject is a pregnant woman. 10.The method of claim 9, wherein the pregnant woman is afflicted with oneor more of gastrointestinal dysbiosis, obesity, metabolic syndrome,gut-mediated systemic inflammation, and leaky gut.
 11. The method of anyof claims 1-8, wherein the subject is an unborn child or newly bornchild, and optionally wherein the mother of said child is afflicted withone or more of gastrointestinal dysbiosis, obesity, metabolic syndrome,gut-mediated systemic inflammation, and leaky gut.
 12. The method of anyof the above claims, wherein the AP-based agent is administered orally.13. The method of any of the above claims, wherein the AP-based agent isIAP selected from human IAP (hIAP), calf IAP (cIAP), and bovine IAP(bIAP), or variants thereof.
 14. The method of any of the above claims,wherein the AP-based agent comprises an amino acid sequence having atleast 95%, or 97%, or 99% sequence identity with any one of SEQ ID NOs:1-11 and 17-18.
 15. The method of any of the above claims, wherein theAP-based agent is bIAP II or a variant thereof.
 16. The method of any ofthe above claims, wherein the AP-based agent comprises an amino sequencehaving at least 95% sequence identity with SEQ ID NO: 2 (bIAP II). 17.The method of any of the above claims, wherein the AP-based agentcomprises an amino sequence having at least 97% sequence identity withSEQ ID NO: 2 (bIAP II).
 18. The method of any of the above claims,wherein the AP-based agent comprises the amino sequence of SEQ ID NO: 2(bIAP II).
 19. The method of claim 7, wherein the AP-based agentcomprises the amino sequence of SEQ ID NO: 2 (bIAP II).
 20. The methodof any of the above claims, wherein the AP-based agent is bIAP IV or avariant thereof.
 21. The method of any of the above claims, wherein theAP-based agent comprises an amino sequence having at least 95% sequenceidentity with SEQ ID NO: 3 (bIAP IV).
 22. The method of any of the aboveclaims, wherein the AP-based agent comprises an amino sequence having atleast 97% sequence identity with SEQ ID NO: 3 (bIAP IV).
 23. The methodof any of the above claims, wherein the AP-based agent comprises theamino sequence of SEQ ID NO: 3 (bIAP IV).
 24. The method of claim 7,wherein the AP-based agent comprises the amino sequence of SEQ ID NO: 3(bIAP IV).
 25. The method of any of the above claims, wherein theAP-based agent comprises a specific activity of at least about 100 U/mgto about 20,000 U/mg.
 26. The method of any of the above claims, whereinthe AP-based agent is stable and/or active in the GI tract, in one ormore of the mouth, esophagus, stomach, duodenum, small intestine,jejunum, ileum, large intestine, colon, cecum, and rectum.
 27. Themethod of any of the above claims, wherein the AP-based agent issubstantially active at a pH of about 6.0 to about
 12. 28. The method ofany of the above claims, wherein the AP-based agent is stable in chyme.29. Use of an AP-based agent for the treatment or prevention of aneurodevelopmental disorder in a subject in need thereof.
 30. The use ofan AP-based agent for the preparation of a medicament for the treatmentor prevention of a neurodevelopmental disorder in a subject in needthereof.
 31. A method of treating or preventing autism spectrum disorder(ASD) in an unborn child or newly born child in need thereof comprising,administering to the mother of said unborn child or newly born child analkaline phosphatase (AP)-based agent, wherein the mother is afflictedwith one or more of gastrointestinal dysbiosis, obesity, metabolicsyndrome, gut-mediated systemic inflammation, and leaky gut, and whereinthe AP-based agent comprises an amino acid sequence having at least 97%sequence identity with SEQ ID NO: 2 or SEQ ID NO: 3.